Recombinant Alpha-Galactosidase A For Treatment Of Fabry Disease

ABSTRACT

Described are compositions comprising α-galactosidase A enzymes with unique carbohydrate profiles, as well as methods for manufacturing and purifying such enzymes. Also described methods of treating, preventing, and/or ameliorating Fabry Disease by administering such enzymes to a subject in need thereof. Also described are compositions comprising migalastat in combination with such α-galactosidase A enzymes.

TECHNICAL FIELD

Principles and embodiments of the present invention relate generally tolysosomal storage disorders, particularly recombinant α-galactosidase Afor the treatment of Fabry disease.

REFERENCE TO THE SEQUENCE LISTING

The Sequence Listing text file submitted herewith, identified as“00712837.TXT” (9 Kb, created Jan. 10, 2018), is hereby incorporated byreference.

BACKGROUND

Fabry disease is a progressive, X-linked inborn error ofglycosphingolipid metabolism caused by a deficiency in the lysosomalenzyme α-galactosidase A (α-Gal A) as a result of mutations in the α-GalA gene (Gla). Despite being an X-linked disorder, females can expressvarying degrees of clinical manifestations. Fabry is a rare disease withincidence estimated between 1 in 40,000 males to 1 in 117,000 in thegeneral population. Moreover, there are variants of later onsetphenotype of Fabry disease that can be under-diagnosed, as they do notpresent with classical signs and symptoms. This, and newborn screeningfor Fabry disease, suggests that the actual incidence of Fabry diseasecan be higher than currently estimated.

Untreated, life expectancy in Fabry patients is reduced and deathusually occurs in the fourth or fifth decade because of vascular diseaseaffecting the kidneys, heart and/or central nervous system. The enzymedeficiency leads to intracellular accumulation of the substrateglobotriaosylceramide (GL-3) in the vascular endothelium and visceraltissues throughout the body. Gradual deterioration of renal function andthe development of azotemia, due to glycosphingolipid deposition,usually occur in the third to fifth decades of life, but can occur asearly as in the second decade. Renal lesions are found in bothhemizygous (male) and heterozygous (female) patients.

Cardiac disease as a result of Fabry disease occurs in most males andmany females. Early cardiac findings include left ventricularenlargement, valvular involvement and conduction abnormalities. Mitralinsufficiency is the most frequent valvular lesion typically present inchildhood or adolescence. Cerebrovascular manifestations resultprimarily from multifocal small-vessel involvement and can includethromboses, transient ischemic attacks, basilar artery ischemia andaneurysm, seizures, hemiplegia, hemianesthesia, aphasia, labyrinthinedisorders, or cerebral hemorrhages. Average age of onset ofcerebrovascular manifestations is 33.8 years. Personality change andpsychotic behavior can manifest with increasing age.

Fabry disease commonly presents with dermatological symptoms, mostcommonly angiokeratoma (small papules that can reside on any region ofthe body). Angiokeratomas appear as dark red or purple skin lesionsranging in size up to several millimeters in diameter. Lesions usuallyappear in adolescence or young adulthood and may increase with age.Other dermatological and soft-tissue related symptoms includeacroparesthesia, abnormal sweating (hypohidrosis and hyperhidrosis) andlymphedema. The presence and extent of cutaneous vascular lesions maycorrelate with the severity of systemic disease.

In addition to dermatological symptoms, patients frequently experienceneuropathy such as burning pain in the extremities(acroparesthesia—often hands and feet). Patients may also experience apain crisis beginning with pain in the extremities and radiating inwardwhich can persist for several days. Neuropathic pain is pain caused bydamage to the somatosensory nervous system. Many types of sensoryreceptors are affected including those in skin, epithelial tissues,skeletal muscles, bones and joints, internal organs, and thecardiovascular system.

The current approved treatment for Fabry disease is enzyme replacementtherapy (“ERT”). Two α-Gal A products are currently available for thetreatment of Fabry disease: agalsidase alfa (Replagal®, Shire HumanGenetic Therapies) and agalsidase beta (Fabrazyme®; Sanofi GenzymeCorporation). These two forms of ERT are intended to compensate for apatient's inadequate α-Gal A activity with a recombinant form of theenzyme, administered intravenously. While ERT is effective in manysettings, the treatment also has limitations. For example, these twoα-Gal A products have not been demonstrated to decrease sufficient riskof stroke, cardiac muscle responds to treatment slowly, and GL-3elimination from some of the cell types of the kidneys is limited.

Accordingly, there remains a need for further improvements for treatingFabry disease. Among others, the present invention includes an improvedrecombinant human alpha-galactosidase A (rhα-Gal A) over the existingERTs and a method of treating a patient in need using the improvedenzyme with a pharmacological chaperone.

SUMMARY

One aspect of the present invention relates to a human recombinantα-galactosidase A (rhα-Gal A).

In various embodiments of this aspect, the rhα-Gal A comprises a proteinhaving at least 98% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2,wherein the rhα-Gal A has at least 25% of total N-linkedoligosaccharides that are mono-mannose-6-phosphate (mono-M6P) and atleast 12% of total N-linked oligosaccharides that arebis-mannose-6-phosphate (bis-M6P). In one or more embodiments, therhα-Gal A has at least 25% of total N-linked oligosaccharides thatcontain sialic acid. In one or more embodiments, the rhα-Gal A has lessthan 20% of total N-linked oligosaccharides that are neutral.

In various embodiments of this aspect, the rhα-Gal A comprises a proteinhaving at least 98% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2,wherein the rhα-Gal A has less than 10% of total N-linkedoligosaccharides that are neutral. In one or more embodiments, therhα-Gal A has at least 50% of total N-linked oligosaccharides thatcontain sialic acid. In one or more embodiments, the rhα-Gal A has atleast 25% of total N-linked oligosaccharides that are mono-M6P and atleast 6% of total N-linked oligosaccharides that are bis-M6P.

In one or more embodiments, the rhα-Gal A has one or more of:

-   -   a. at least 17% of total N-linked oligosaccharides that contain        a single sialic acid residue;    -   b. at least 20% of total N-linked oligosaccharides that contain        two sialic acid residues;    -   c. at least 40% of total N-linked oligosaccharides that contain        one or two sialic acid residues; or    -   d. at least 6 moles of sialic acid residues per mole of rhα-Gal        A homodimer.

In one or more embodiments, the rhα-Gal A has two, three, or four of:

-   -   a. at least 17% of total N-linked oligosaccharides that contain        a single sialic acid residue;    -   b. at least 20% of total N-linked oligosaccharides that contain        two sialic acid residues;    -   c. at least 40% of total N-linked oligosaccharides that contain        one or two sialic acid residues; or    -   d. at least 6 moles of sialic acid residues per mole of rhα-Gal        A homodimer.

In one or more embodiments, the rhα-Gal A has at least 7 moles of sialicacid residues per mole of rhα-Gal A homodimer.

In one or more embodiments, the rhα-Gal A has at least 22% of totalN-linked oligosaccharides that contain two sialic acid residues.

In one or more embodiments, the rhα-Gal A has at least 14% of totalN-linked oligosaccharides that are bis-mannose-6-phosphate.

Another aspect of the present invention relates to a method of producinga recombinant protein product comprising rhα-Gal A. In variousembodiments of this aspect, the method comprises culturing Chinesehamster ovary (CHO) host cells in a bioreactor that secrete rhα-Gal A,wherein the rhα-Gal A comprises a protein having at least 98% sequenceidentity to SEQ ID NO: 1 or SEQ ID NO: 2; removing media from thebioreactor; filtering the media to provide a filtrate; loading thefiltrate onto an anion exchange chromatography (AEX) column to capturethe rhα-Gal A; and eluting a first protein product comprising therhα-Gal A from the AEX column. In one or more embodiments, the firstprotein product is an intermediate protein product that is subjected tofurther processing and/or purification before becoming a finalizedprotein product (e.g. suitable to use as an ERT). In other embodiments,the first protein product is a finalized protein product.

Another aspect of the present invention relates to recombinant proteinproduct made by the processes described herein.

Another aspect of the present invention relates to a method of treatingFabry disease, wherein a patient in need thereof is administered therhα-Gal A as described herein or a recombinant protein productcomprising rhα-Gal A as described herein.

Another aspect of the present invention relates to a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and therhα-Gal A as described herein or a recombinant protein productcomprising rhα-Gal A as described herein. In one or more embodiments,the pharmaceutical composition comprises about 0.5 to about 20 μMrhα-Gal A and about 50 to about 20,000 μM migalastat or salt thereof. Inone or more embodiments, the pharmaceutical composition comprises about1 to about 10 μM rhα-Gal A and about 100 to about 10,000 μM migalastator salt thereof. In one or more embodiments, the migalastat and rhα-GalA are present in a molar ratio of migalastat to α-galactosidase A ofbetween about 13,000:1 and about 50:1.

Another aspect of the present invention relates to a method of treatingFabry disease, the method comprising administering a pharmaceuticalcomposition as described herein. In one or more embodiments, the rhα-GalA is administered at a dose of about 0.5 mg/kg to about 10 mg/kg.

In one or more embodiments, the patient is co-administered apharmacological chaperone for α-Gal A within 4 hours of theadministration of the rhα-Gal A or the pharmaceutical compositioncomprising the recombinant protein product. In one or more embodiments,the pharmacological chaperone comprises migalastat or salt thereof.

In one or more embodiments, the pharmacological chaperone isadministered orally and pharmaceutical composition comprising rhα-Gal Ais administered intravenously.

In one or more embodiments, the pharmacological chaperone isco-formulated with the rhα-Gal A. In one or more embodiments, themigalastat or salt thereof is administered at a dose of about 1 mg/kg toabout 100 mg/kg.

In one or more embodiments, the pharmaceutical composition comprisingrhα-Gal A (and optionally a pharmacological chaperone such asmigalastat) is administered once a month to once a week. In one or moreembodiments, the pharmaceutical composition is administered every otherweek.

Another aspect of the present invention relates to a method for treatingFabry disease, the method comprising administering a pharmaceuticalcomposition comprising rhα-Gal A as described herein and optionally apharmacological chaperone such as migalastat to a patient in needthereof.

In one or more embodiments, the patient is co-administered apharmacological chaperone for α-Gal A within 4 hours of theadministration of the pharmaceutical composition comprising rhα-Gal A.

In one or more embodiments, the pharmacological chaperone isadministered orally and pharmaceutical composition comprising rhα-Gal Ais administered intravenously.

In one or more embodiments, the migalastat or salt thereof isadministered at a dose of about 1 mg/kg to about 100 mg/kg.

In one or more embodiments, the pharmaceutical composition isadministered once a month to once a week.

In one or more embodiments, the pharmaceutical composition isadministered every other week.

In one or more embodiments, the administering is contacting a cell withthe rhα-Gal A bound to the pharmacological chaperone.

In one or more embodiments, the cell is located in the patient's heart.

In one or more embodiments, the cell is located in the patient's kidney.

In one or more embodiments, the cell is located in the patient's skin

In one or more embodiments, the pharmacological chaperone is migalastathydrochloride.

Another aspect of the present invention relates to a method of enhancingthe activity level of α-galactosidase-A protein in a lysosome in amammalian cell, the method comprising contacting the mammalian cell withrhα-Gal A as described herein, wherein the rhα-Gal A is optionally boundto a pharmacological chaperone.

In one or more embodiments, the rhα-Gal A is administered at a dose ofabout 0.5 mg/kg to about 10 mg/kg.

In one or more embodiments, the pharmacological chaperone isco-formulated with the rhα-Gal A.

In one or more embodiments, the patient is co-administered apharmacological chaperone for α-Gal A within 4 hours of theadministration of the pharmaceutical composition comprising rhα-Gal A.

In one or more embodiments, the pharmacological chaperone isadministered orally and pharmaceutical composition comprising rhα-Gal Ais administered intravenously.

In one or more embodiments, the migalastat or salt thereof isadministered at a dose of about 1 mg/kg to about 100 mg/kg.

In one or more embodiments, the pharmaceutical composition isadministered once a month to once a week.

In one or more embodiments, the pharmaceutical composition isadministered every other week.

In one or more embodiments, the cell is a heart cell.

In one or more embodiments, the cell is a kidney cell.

In one or more embodiments, the cell is a skin cell.

In one or more embodiments, the contacting is performed byadministration of the rhα-Gal A and the pharmacological chaperone.

In one or more embodiments, the administering is systemic administrationof both the pharmacological chaperone and the rhα-Gal A.

In one or more embodiments, both the pharmacological chaperone and therhα-Gal A are co-administered.

In one or more embodiments, the pharmacological chaperone and therhα-Gal A are present in a composition formulated for intravenous,intraarterial, intramuscular, intradermal, subcutaneous, orintraperitoneal administration.

In one or more embodiments, the composition is formulated forintravenous administration.

In one or more embodiments, the pharmacological chaperone is migalastator salt thereof.

In one or more embodiments, the pharmaceutical composition comprises:

about 0.5 to about 20 μM rhα-Gal A; and

about 50 to about 20,000 μM migalastat or salt thereof.

In one or more embodiments, the pharmaceutical composition comprises:

about 1 to about 10 μM rhα-Gal A; and

about 100 to about 10,000 μM migalastat or salt thereof.

In one or more embodiments, the ratio of migalastat or salt thereof toα-galactosidase A of between about 13,000:1 and about 50:1.

In one or more embodiments, the cell is in vitro.

In one or more embodiments, the cell is a human cell.

In one or more embodiments, the cell is in a subject.

In one or more embodiments, the subject is a patient in need.

In one or more embodiments, the cell is located in the subject's heart,kidney or skin.

In one or more embodiments, the cell is located in the subject's heart.

In one or more embodiments, the cell is located in the subject's kidney.

In one or more embodiments, the cell is located in the subject's skin.

In one or more embodiments, the patient has been diagnosed as havingFabry disease.

Another aspect of the present invention relates to a method of reducingthe level of GL-3 in an organ of a patient in need, the methodcomprising administering to the patient a composition comprising atherapeutically effective amount of (i) a pharmacological chaperone and(ii) rhα-Gal A as described herein.

In one or more embodiments, the administering is performed byco-administration.

In one or more embodiments, the administering is systemic administrationof both the pharmacological chaperone and the rhα-Gal A.

In one or more embodiments, the composition is formulated forintravenous, intraarterial, intramuscular, intradermal, subcutaneous, orintraperitoneal administration.

In one or more embodiments, the composition is formulated forintravenous administration.

In one or more embodiments, the pharmacological chaperone is migalastator salt thereof.

In one or more embodiments, the pharmaceutical composition comprises:

about 0.5 to about 20 μM rhα-Gal A; and

about 50 to about 20,000 μM migalastat or salt thereof.

In one or more embodiments, the pharmaceutical composition comprises:

about 1 to about 10 μM rhα-Gal A; and

about 100 to about 10,000 μM migalastat or salt thereof.

In one or more embodiments, the ratio of migalastat or salt thereof toα-galactosidase A of between about 13,000:1 and about 50:1.

In one or more embodiments, the organ is heart, kidney or skin.

In one or more embodiments, the organ is heart.

In one or more embodiments, the organ is kidney.

In one or more embodiments, the organ is skin.

In one or more embodiments, the subject has been diagnosed as havingFabry disease.

Another aspect of the present invention relates to a method of treatingFabry disease, the method comprising contacting a mammalian cell with aneffective amount of rhα-Gal A, wherein contacting the cell with therhα-Gal A provides a greater reduction in GL-3 than contacting withFabrazyme (agalsidase beta).

In one or more embodiments, the contacting is administering to a subjectan effective amount of the rhα-Gal A.

In one or more embodiments, the cell is in a subject.

In one or more embodiments, the subject is a patient in need.

In one or more embodiments, the reduction in GL-3 is measured in hearttissue.

In one or more embodiments, the reduction in GL-3 is measured in kidneytissue.

In one or more embodiments, the reduction in GL-3 is measured in skintissue.

In one or more embodiments, the rhα-Gal A is bound to a pharmacologicalchaperone.

In one or more embodiments, the pharmacological chaperone is migalastator a salt thereof.

In one or more embodiments, the migalastat or salt thereof isadministered at a dose of 3 mg/kg.

In one or more embodiments, the migalastat or salt thereof isadministered at a dose of 10 mg/kg.

In one or more embodiments, the migalastat or salt thereof isadministered at a dose of 30 mg/kg.

In one or more embodiments, the migalastat or salt thereof isadministered at a dose of 300 mg/kg.

In one or more embodiments, the rhα-Gal A and the pharmacologicalchaperone is co-administered.

In one or more embodiments, the rhα-Gal A and the pharmacologicalchaperone is co-formulated.

In one or more embodiments, a dose of 1 mg/kg rhα-Gal A provides agreater reduction in GL-3 than administration of Fabrazyme (agalsidasebeta) to Gla knockout mice at a dose of 1 mg/kg.

In one or more embodiments, a dose of 10 mg/kg rhα-Gal A provides agreater reduction in GL-3 than administration of Fabrazyme (agalsidasebeta) to Gla knockout mice at a dose of 10 mg/kg.

In one or more embodiments, the reduction in GL-3 after administrationof the pharmaceutical composition to Gla knockout mice is at least 10%greater than the reduction in GL-3 after administration of Fabrazyme(agalsidase beta).

In one or more embodiments, the reduction in GL-3 after administrationof the pharmaceutical composition to Gla knockout mice is at least 20%greater than the reduction in GL-3 after administration of Fabrazyme(agalsidase beta).

Another aspect of the present invention relates to a method of treatingFabry disease, the method comprising contacting a mammalian cell with aneffective amount of rhα-Gal A, wherein contacting the cell with therhα-Gal A provides a greater reduction in plasma lyso-Gb3 thancontacting with Fabrazyme (agalsidase beta).

In one or more embodiments, the contacting is administering an effectiveto a subject an effective amount of the rhα-Gal A.

In one or more embodiments, the cell is in a subject.

In one or more embodiments, the subject is a patient in need.

In one or more embodiments, the reduction in plasma lyso-Gb3 is measuredin heart tissue.

In one or more embodiments, the reduction in plasma lyso-Gb3 is measuredin kidney tissue.

In one or more embodiments, the reduction in plasma lyso-Gb3 is measuredin skin tissue.

In one or more embodiments, the rhα-Gal A is bound to a pharmacologicalchaperone.

In one or more embodiments, the pharmacological chaperone is migalastator a salt thereof.

In one or more embodiments, the migalastat or salt thereof isadministered at a dose of 3 mg/kg.

In one or more embodiments, the migalastat or salt thereof isadministered at a dose of 10 mg/kg.

In one or more embodiments, the migalastat or salt thereof isadministered at a dose of 30 mg/kg.

In one or more embodiments, the migalastat or salt thereof isadministered at a dose of 300 mg/kg.

In one or more embodiments, the rhα-Gal A and the pharmacologicalchaperone is co-administered.

In one or more embodiments, the rhα-Gal A and the pharmacologicalchaperone is co-formulated.

In one or more embodiments, a dose of 1 mg/kg rhα-Gal A provides agreater reduction in plasma lyso-Gb3 than administration of Fabrazyme(agalsidase beta) to Gla knockout mice at a dose of 1 mg/kg.

In one or more embodiments, a dose of 10 mg/kg rhα-Gal A provides agreater reduction in plasma lyso-Gb3 than administration of Fabrazyme(agalsidase beta) to Gla knockout mice at a dose of In one or moreembodiments, the reduction in plasma lyso-Gb3 after administration ofthe pharmaceutical composition to Gla knockout mice is at least 10%greater than the reduction in plasma lyso-Gb3 after administration ofFabrazyme (agalsidase beta).

In one or more embodiments, the reduction in plasma lyso-Gb3 afteradministration of the pharmaceutical composition to Gla knockout mice isat least 20% greater than the reduction in plasma lyso-Gb3 afteradministration of Fabrazyme (agalsidase beta).

Another aspect of the present invention relates to a method of treatingFabry disease, the method comprising contacting a mammalian cell with aneffective amount rhα-Gal A), wherein contacting the cell with therhα-Gal A provides a greater reduction in one or more substrates thancontacting with Fabrazyme (agalsidase beta).

In one or more embodiments, the one or more substrates comprises GL-3 orplasma lyso-Gb3.

In one or more embodiments, the one or more substrates is GL-3.

In one or more embodiments, the one or more substrates is plasmalyso-Gb3.

In one or more embodiments, the rhα-Gal A is bound to a pharmacologicalchaperone.

In one or more embodiments, the pharmacological chaperone is migalastator a salt thereof.

In one or more embodiments, the migalastat or salt thereof isadministered at a dose of 3 mg/kg.

In one or more embodiments, the migalastat or salt thereof isadministered at a dose of 10 mg/kg.

In one or more embodiments, the migalastat or salt thereof isadministered at a dose of 30 mg/kg.

In one or more embodiments, the migalastat or salt thereof isadministered at a dose of 300 mg/kg

BRIEF DESCRIPTION OF THE DRAWINGS

Further features of the present invention will become apparent from thefollowing written description and the accompanying figures, in which:

FIG. 1A shows non-phosphorylated high mannose glycan, a mono-M6P glycan,and a bis-M6P glycan;

FIG. 1B shows the chemical structure of the M6P group;

FIG. 2A shows productive targeting of rhα-Gal A via glycans bearing M6Pto target tissues;

FIG. 2B shows non-productive drug clearance to non-target tissues (e.g.liver and spleen) or by binding of non-M6P glycans to non-targettissues;

FIG. 3 shows the dual expression vectors used to transfect a suspensionCHO-K1 culture;

FIG. 4 shows density of viable cells from CHO cell pools transformedwith plasmid expressing rhα-Gal A;

FIG. 5 shows the degree of enzyme activity produced by each cell poolover time;

FIGS. 6A-6F show the degree of CIMPR bound rhα-Gal A in each cell-poolculture supernatant at day 15-17;

FIGS. 7A-7F show CIMPR profiles of Fabrazyme and rhα-Gal A of differentcell line and different growth conditions;

FIGS. 8A-8B show increased thermostability of rhα-Gal A in the presenceof migalastat;

FIG. 9 shows the extent of glycosylation removal by PNGase as analyzedby SDS-PAGE;

FIGS. 10A-10F show the glycan analysis of Fabrazyme and rhα-Gal A ofdifferent cell lines and different growth conditions;

FIG. 11 shows a glycan comparison of Fabrazyme and rhα-Gal A ofdifferent cell lines and different growth conditions;

FIG. 12 shows the pharmacokinetics of Fabrazyme and two clones ofrhα-Gal A in the presence and absence of migalastat;

FIGS. 13A-13C show α-Gal A activity in Gla KO mice heart (13A), kidney(13B) and skin (13C) one day after a single administration of variousERTs with and without migalastat;

FIGS. 14A-14D show α-Gal A activity in Gla KO mice heart (14A), kidney(14B), skin (14C) and liver (14D) seven days after a singleadministration of various ERTs with and without migalastat;

FIGS. 15A-15D show GL-3 levels in Gla KO mice heart (15A), kidney (15B),and skin (15C) and plasma lyso-Gb3 (15D) after a single administrationof various ERTs with and without migalastat;

FIGS. 16A and 16B show the pharmacokinetics of Fabrazyme and rhα-Gal Aproduced by different processes;

FIGS. 17A and 17B show GL-3 levels in Gla KO mice heart (17A) and kidney(17B) after a single administration of various ERTs with and withoutmigalastat;

FIGS. 18A-18F show the pharmacokinetics of Fabrazyme and rhα-Gal A atvarious doses with and without migalastat;

FIGS. 19A-19D show GL-3 levels in Gla KO mice heart (19A), kidney (19B),and skin (19C) and plasma lyso-Gb3 (19D) after repeat administrations ofvarious ERTs with and without migalastat;

FIGS. 20A-20C show α-Gal A activity in Gla KO mice heart (20A), kidney(20B) and skin (20C) after repeat administrations of various ERTs withand without migalastat;

FIGS. 21A-21D shows GL-3 levels in Gla KO mice heart (21A), kidney(21B), skin (21C) and plasma lyso-Gb3 (21D) after a singleadministration of various ERTs with and without migalastat;

FIG. 22 shows a single dose cohort design for a study of migalastatintravenous dosing in human subjects;

FIG. 23 shows cohort crossover design for a study of migalastatintravenous and oral administration in human subjects;

FIGS. 24A and 24B show the pharmacokinetic profile of migalastatintravenous dosing in human subjects; and

FIGS. 25A and 25B show a comparison of the pharmacokinetic profiles ofintravenous and oral administration of migalastat.

DETAILED DESCRIPTION

Before describing several exemplary embodiments of the invention, it isto be understood that the invention is not limited to the details ofconstruction or process steps set forth in the following description.The invention is capable of other embodiments and of being practiced orbeing carried out in various ways.

Various aspects of the invention pertain to novel recombinant humanα-galactosidase A (rhα-Gal A). Other aspects of the invention pertain torecombinant proteins produced by the processes described herein, as wellas pharmaceutical compositions, methods of treatment, and uses of suchrecombinant proteins.

Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner.

In the present specification, except where the context requiresotherwise due to express language or necessary implication, the word“comprises”, or variations such as “comprises” or “comprising” is usedin an inclusive sense i.e. to specify the presence of the statedfeatures but not to preclude the presence or addition of furtherfeatures in various embodiments of the invention.

As used herein, the term “Fabry disease,” (also referred to asalpha-galactosidase A deficiency; Anderson-Fabry disease; angiokeratomacorporis diffusum; angiokeratoma diffuse; ceramide trihexosidasedeficiency; GLA deficiency, and hereditary dystopic lipidosis) isintended to refer to a genetic lysosomal storage disorder characterizedby mutations in the Gla gene which codes for human alpha-galactosidaseA. The term includes all stages and forms of the disease experienced byinfantile, juvenile, and adult patients.

As used here the term “alpha-galactosidase A” (also known as α-Gal A;E.C. 3.2.1.22 family of alpha-galactosidases) is intended to refer to alysosomal enzyme which breaks down alpha-galactosides in the lysosome.Human α-galactosidase is a homodimeric glycoprotein that hydrolyses theterminal alpha-galactosyl moieties from glycolipids and glycoproteins.The enzyme predominantly hydrolyzes globotriaosylceramide (“GL-3”, alsoknown as Gb3 or ceramide trihexoside), and can catalyze the hydrolysisof melibiose into galactose and glucose. Another substrate of the enzymeis plasma globotriaosylsphingosine (“plasma lyso-Gb3”). A variety ofmutations in the gene affect the synthesis, processing, and stability ofthe enzyme which cause Fabry disease as a result of a failure tocatabolize alpha-D-galactosyl glycolipid moieties. Alpha-galactosidase Acatalyzes the removal of terminal α-galactose residues frompolysaccharides, glycolipids, and glycopeptides. The Gla gene (NationalCenter for Biotechnology Information (NCBI) Gene ID 2717) encodes forhuman alpha-galactosidase A and has been mapped to the long arm of the Xchromosome at position 22. As used herein the abbreviation “α-Gal A” isintended to refer to the alpha-galactosidase A enzyme. The italicized“Gla” is intended to refer to the human gene coding for human α-Gal A.The abbreviation rhα-Gal A is intended to refer to recombinant humanα-Gal A. There are currently more than 400 mutations identified in Fabrypatients. The most common form of mutation leads to a single amino acidchange in the enzyme. Other types of mutations include deletions,insertions, premature stop codons, frame-shift mutations, and splicesite mutations.

The term “agalsidase beta” is intended to refer to a homodimericrecombinant human α-Gal A marketed by Genzyme under the name Fabrazyme®.Fabrazyme (agalsidase beta) has a molecular weight of approximately 100kDa with a recommended dosage of 1.0 mg/kg body weight infused every twoweeks as an IV infusion. The initial infusion rate is recommended to beno more than 0.25 mg/min (15 mg/hr). Infusion reactions to Fabrazyme(agalsidase beta), some of which are severe, have occurred in Fabrypatients. Infusion reactions include fever, rigors, chest tightness,hypertension, hypotension, pruritis, myalgia, dyspnea, urticarial,abdominal pain, and headache. It is suggested that patients be givenantipyretic prior to infusion.

The term “agalsidase alfa” is intended to refer to a homodimericrecombinant human α-Gal A marketed by Shire Human Genetic Therapies Inc.under the name Replagal®. Replagal (agalsidase alfa) is a homodimer ofapproximately 100 kDa with each subunit containing 398 amino acidresidues with an identical amino acid sequence to wild-type human α-GalA. The recommended dose of Replagal is 0.2 mg/kg every other week byintravenous infusion.

The term “rhα-Gal A” is intended to refer to a recombinant humanalpha-galactosidase A as described herein and as exemplified in theExamples below.

As used herein, the term “glycan” or “oligosaccharide” is intended torefer to a polysaccharide chain covalently bound to an amino acidresidue on a protein or polypeptide. As used herein, the term“N-glycan”, “N-oligosaccharide” “N-linked glycan” or “N-linkedoligosaccharide” is intended to refer to a polysaccharide chain attachedto an amino acid residue on a protein or polypeptide through covalentbinding to a nitrogen atom of the amino acid residue. For example, anN-glycan can be covalently bound to the side chain nitrogen atom of anasparagine residue. Glycans can contain one or several monosaccharideunits, and the monosaccharide units can be covalently linked to form astraight chain or a branched chain. In at least one embodiment, N-glycanunits attached to rhα-Gal A can comprise one or more monosaccharideunits each independently selected from N-acetylglucosamine, mannose,galactose or sialic acid. The N-glycan units on the protein can bedetermined by any appropriate analytical technique, such as massspectrometry. In some embodiments, the N-glycan units can be determinedby liquid chromatography-tandem mass spectrometry (LC-MS/MS) utilizingan instrument such as the Thermo Scientific Orbitrap Velos Pro™ MassSpectrometer, Thermo Scientific Orbitrap Fusion Lumos Tribid™ MassSpectrometer or Waters Xevo® G2-XS QTof Mass Spectrometer.

As used herein, the term “high-mannose N-glycan” is intended to refer toan N-glycan having one to six or more mannose units. In at least oneembodiment, a high mannose N-glycan unit can contain abis(N-acetylglucosamine) chain bonded to an asparagine residue andfurther bonded to a branched polymannose chain. As used hereininterchangeably, the term “M6P” or “mannose-6-phosphate” is intended torefer to a mannose unit phosphorylated at the 6 position; i.e. having aphosphate group bonded to the hydroxyl group at the 6 position. In atleast one embodiment, one or more mannose units of one or more N-glycanunits are phosphorylated at the 6 position to form mannose-6-phosphateunits. In at least one embodiment, the term “M6P” or“mannose-6-phosphate” refers to both a mannose phosphodiester havingN-acetylglucosamine (GlcNAc) as a “cap” on the phosphate group, as wellas a mannose unit having an exposed phosphate group lacking the GlcNAccap. In at least one embodiment, the N-glycans of a protein can havemultiple M6P groups, with at least one M6P group having a GlcNAc cap andat least one other M6P group lacking a GlcNAc cap.

As used herein, the term “complex N-glycan” is intended to refer to anN-glycan containing one or more N-acetylglucosamine,N-acetylgalactosamine, galactose, fucose and/or sialic acid units. In atleast one embodiment, a complex N-glycan can be a high-mannose N-glycanin which one or mannose units are further bonded to one or moremonosaccharide units each independently selected fromN-acetylglucosamine, N-acetylgalactosamine, galactose, fucose and sialicacid.

As used herein, the compound migalastat, also known as1-deoxygalactonojirimycin (1-DGJ), (2R, 3S, 4R,5S0-2-(hydroxymethyl)piperidine-3,4,5-triol and is a compound having thefollowing chemical formula:

As discussed below, pharmaceutically acceptable salts of migalastat mayalso be used in the present invention. When a salt of migalastat isused, the dosage of the salt will be adjusted so that the dose ofmigalastat received by the patient is equivalent to the amount whichwould have been received had the migalastat free base been used. Oneexample of a pharmaceutically acceptable salt of migalastat ismigalastat HCl:

The term “migalastat” encompasses migalastat free base or apharmaceutically acceptable salt thereof (e.g., migalastat HCl as shownabove), unless specifically indicated to the contrary.

As used herein, the term “pharmacological chaperone” or sometimes simplythe term “chaperone” is intended to refer to a molecule thatspecifically binds to α-Gal A and has one or more of the followingeffects: enhances the formation of a stable molecular conformation ofthe protein; enhances proper trafficking of the protein from theendoplasmic reticulum to another cellular location, preferably a nativecellular location, so as to prevent endoplasmic reticulum-associateddegradation of the protein; prevents aggregation of conformationallyunstable or misfolded proteins; restores and/or enhances at leastpartial wild-type function, stability, and/or activity of the protein;and/or improves the phenotype or function of the cell harboring α-Gal A.Thus, a pharmacological chaperone for α-Gal A is a molecule that bindsto α-Gal A, resulting in proper folding, trafficking, non-aggregation,and activity of α-Gal A. As used herein, this term includes but is notlimited to active site-specific chaperones (ASSCs) which bind in theactive site of the enzyme, inhibitors or antagonists, and agonists. Inat least one embodiment, the pharmacological chaperone can be aninhibitor or antagonist of α-Gal A. As used herein, the term“antagonist” is intended to refer to any molecule that binds to α-Gal Aand either partially or completely blocks, inhibits, reduces, orneutralizes an activity of α-Gal A. In at least one embodiment, thepharmacological chaperone is migalastat. Another non-limiting example ofa pharmacological chaperone for α-Gal A isN-butyldeoxygalactonojirimycin (NB-DGJ) or a salt thereof.

As used herein, the term “active site” is intended to refer to a regionof a protein that is associated with and necessary for a specificbiological activity of the protein. In at least one embodiment, theactive site can be a site that binds a substrate or other bindingpartner and contributes the amino acid residues that directlyparticipate in the making and breaking of chemical bonds. Active sitesin this invention can encompass catalytic sites of enzymes, antigenbinding sites of antibodies, ligand binding domains of receptors,binding domains of regulators, or receptor binding domains of secretedproteins. The active sites can also encompass transactivation,protein-protein interaction, or DNA binding domains of transcriptionfactors and regulators.

As used herein, the term “AUC” is intended to refer to a mathematicalcalculation to evaluate the body's total exposure over time to a givendrug. In a graph plotting how concentration in the blood of a drugadministered to a subject changes with time after dosing, the drugconcentration variable lies on the y-axis and time lies on the x-axis.The area between the drug concentration curve and the x-axis for adesignated time interval is the AUC (“area under the curve”). AUCs areused as a guide for dosing schedules and to compare the bioavailabilityof different drugs' availability in the body.

As used herein, the term “Cmax” is intended to refer to the maximumplasma concentration of a drug achieved after administration to asubject.

As used herein, the term “t_(max)” is intended to refer to the time ofthe maximum plasma concentration of a drug achieved after administrationto a subject.

As used herein, the term “t½” is intended to refer to the terminalelimination half-life.

As used herein, the term “volume of distribution” or “V” is intended torefer to the theoretical volume that would be necessary to contain thetotal amount of an administered drug at the same concentration that itis observed in the blood plasma, and represents the degree to which adrug is distributed in body tissue rather than the plasma. Higher valuesof V indicate a greater degree of tissue distribution. “Central volumeof distribution” or “V_(c)” is intended to refer to the volume ofdistribution within the blood and tissues highly perfused by blood.“Peripheral volume of distribution” or “V2” is intended to refer to thevolume of distribution within the peripheral tissue.

As used interchangeably herein, the terms “clearance”, “systemicclearance” or “CL” are intended to refer to the volume of plasma that iscompletely cleared of an administered drug per unit time. “Peripheralclearance” is intended to refer to the volume of peripheral tissue thatis cleared of an administered drug per unit time.

As used herein, the “therapeutically effective dose” and “effectiveamount” are intended to refer to an amount of α-galactosidase A and/orof a pharmacological chaperone (e.g., migalastat or a salt thereof)and/or of a combination thereof, which is sufficient to result in atherapeutic response in a subject. A therapeutic response may be anyresponse that a user (for example, a clinician) will recognize as aneffective response to the therapy, including any surrogate clinicalmarkers or symptoms described herein and known in the art. Thus, in atleast one embodiment, a therapeutic response can be an amelioration orinhibition of one or more symptoms or markers of Fabry disease such asthose known in the art. Symptoms or markers of Fabry disease include butare not limited to decreased α-galactosidase A tissue activity;cloudiness of the cornea, burning sensations in hands and feet, skinblemishes (reddish-purple raised lesions), gastrointestinal problems,frequent bowel movements shortly after eating, telangiectasia, painattacks, autonomic dysfunction, angina, EKG changes, paresthesia,lymphedema, skin lesions, angiokeratomas, corneal opacities,hypertension, renal failure, acroparesthesia, hypohidrosis, transientischemic attacks, stroke, muscle weakness, hemiparesis, vertigo, hearingloss, tinnitus, nystagmus, head pain, hemiataxia, ataxia, leg swelling,cataracts, chronic airflow obstructions, dyspnea, coronary heartdisease, myocardial infarction, arrhythmias, episodic diarrhea, nausea,vomiting, protein urea. It should be noted that a concentration ofmigalastat that has an inhibitory effect on α-galactosidase A mayconstitute an “effective amount” for purposes of this invention becauseof dilution (and consequent shift in binding due to the change inequilibrium), bioavailability and metabolism of migalastat uponadministration in vivo.

As used herein, the term “enzyme replacement therapy” or “ERT” isintended to refer to the introduction of a non-native, purified enzymeinto an individual having a deficiency in such enzyme. The administeredprotein can be obtained from natural sources or by recombinantexpression. The term also refers to the introduction of a purifiedenzyme in an individual otherwise requiring or benefiting fromadministration of a purified enzyme. In at least one embodiment, such anindividual suffers from enzyme insufficiency. The introduced enzyme maybe a purified, recombinant enzyme produced in vitro, or a proteinpurified from isolated tissue or fluid, such as, for example, placentaor animal milk, or from plants.

As used herein, the term “combination therapy” is intended to refer toany therapy wherein two or more individual therapies are administeredconcurrently or consecutively. In at least one embodiment, the resultsof the combination therapy are enhanced as compared to the effect ofeach therapy when it is performed individually. Enhancement may includeany improvement of the effect of the various therapies that may resultin an advantageous result as compared to the results achieved by thetherapies when performed alone. Enhanced effect or results can include asynergistic enhancement, wherein the enhanced effect is more than theadditive effects of each therapy when performed by itself; an additiveenhancement, wherein the enhanced effect is substantially equal to theadditive effect of each therapy when performed by itself; or less than asynergistic effect, wherein the enhanced effect is lower than theadditive effect of each therapy when performed by itself, but stillbetter than the effect of each therapy when performed by itself.Enhanced effect may be measured by any means known in the art by whichtreatment efficacy or outcome can be measured. An enhanced effect caninclude a lessening in the frequency or severity of side-effects or offtarget activity of the therapeutic.

As used herein, the term “pharmaceutically acceptable” is intended torefer to molecular entities and compositions that are physiologicallytolerable and do not typically produce untoward reactions whenadministered to a human. Preferably, as used herein, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans.

As used herein, the term “carrier” is intended to refer to a diluent,adjuvant, excipient, or vehicle with which a compound is administered.Suitable pharmaceutical carriers are known in the art and, in at leastone embodiment, are described in “Remington's Pharmaceutical Sciences”by E. W. Martin, 18th Edition, or other editions.

As used herein, the terms “subject” or “patient” are intended to referto a human or non-human animal. In at least one embodiment, the subjectis a mammal. In at least one embodiment, the subject is a human.

As used herein, the term “anti-drug antibody” is intended to refer to anantibody specifically binding to a drug administered to a subject andgenerated by the subject as at least part of a humoral immune responseto administration of the drug to the subject. In at least one embodimentthe drug is a therapeutic protein drug product. The presence of theanti-drug antibody in the subject can cause immune responses rangingfrom mild to severe, including but not limited to life-threateningimmune responses which include but are not limited to anaphylaxis,cytokine release syndrome and cross-reactive neutralization ofendogenous proteins mediating critical functions. In addition oralternatively, the presence of the anti-drug antibody in the subject candecrease the efficacy of the drug.

As used herein, the terms “about” and “approximately” are intended torefer to an acceptable degree of error for the quantity measured giventhe nature or precision of the measurements. For example, the degree oferror can be indicated by the number of significant figures provided forthe measurement, as is understood in the art, and includes but is notlimited to a variation of ±1 in the most precise significant figurereported for the measurement. Typical exemplary degrees of error arewithin 20 percent (%), preferably within 10%, and more preferably within5% of a given value or range of values. Alternatively, and particularlyin biological systems, the terms “about” and “approximately” can meanvalues that are within an order of magnitude, preferably within 5-foldand more preferably within 2-fold of a given value. Numerical quantitiesgiven herein are approximate unless stated otherwise, meaning that theterm “about” or “approximately” can be inferred when not expresslystated.

The term “concurrently” as used herein is intended to mean at the sametime as or within a reasonably short period of time before or after, aswill be understood by those skilled in the art. For example, if twotreatments are administered concurrently with each other, one treatmentcan be administered before or after the other treatment, to allow fortime needed to prepare for the later of the two treatments. Therefore“concurrent administration” of two treatments includes but is notlimited to one treatment following the other by 20 minutes or less,about 20 minutes, about 15 minutes, about 10 minutes, about 5 minutes,about 2 minutes, about 1 minute or less than 1 minute.

Recombinant Human α-Galactosidase A

Various embodiments of the present invention relate to recombinant humanα-galactosidase A (rhα-Gal A) having unique carbohydrate profiles.Lysosomal enzyme replacement therapies generally rely on the binding ofthe enzyme to the cation independent mannose-6-phosphate receptor(CIMPR). Specifically, specific carbohydrates have affinity for theCIMPR, most notably M6P and particularly bis-M6P. Upon binding the CIMPRon the surface of a cell the enzyme is internalized and trafficked tothe lysosome. The lysosome is the primary site of substrate accumulationand ultimately the site where enzyme is required to degrade thesubstrate.

There are four potential N-linked glycosylation sites on each chain ofthe rhα-Gal A homodimer, three of which are typically glycosylated.Since each glycosylation site is heterogeneous in the type of N-linkedoligosaccharides (N-glycans) present, rhα-Gal A consists of a complexmixture of proteins with N-glycans having varying binding affinities forcarbohydrate receptors. The rhα-Gal A that contains high mannoseN-glycans having one M6P group (mono-M6P) binds to CIMPR with low(˜7,000 nM) affinity while rhα-Gal A that contains two M6P groups on thesame N-glycan (bis-M6P) bind with high (˜2 nM) affinity. Representativestructures for non-phosphorylated, mono-M6P, and bis-M6P glycans areshown by FIG. 1A. The M6P group is shown by FIG. 1B. Productive drugtargeting is shown in FIG. 2A.

Recombinant proteins that do not have phosphorylated N-glycans lackaffinity for the CIMPR. Non-phosphorylated high mannose glycans can alsobe cleared by the mannose receptor which results in nonproductiveclearance of the ERT (FIG. 2B).

The other type of N-glycans, complex carbohydrates, which containgalactose and sialic acids, are also present on rhα-Gal A. Since complexN-glycans are not phosphorylated they have no affinity for CIMPR.Complex-type N-glycans with exposed galactose residues have moderate tohigh affinity for the asialoglycoprotein receptor on liver hepatocyteswhich leads to rapid non-productive clearance of rhα-Gal A (FIG. 2B).Asialoglycoprotein receptors and mannose receptors are highly expressedin liver cells and remove proteins from circulation that have exposedgalactose and mannose (among others). One way to avoid thisnon-productive pathway of enzyme absorption through theasialoglycoprotein receptor is to block terminal sites with sialic acid.Proteins with terminal sialic acid are not subject to thisnon-productive pathway of removal and are thus available for targetingto the CIMPR.

Due to the inefficiency of delivering conventional enzyme replacementtherapies to lysosomes, such therapies are often associated withproblems, including generation of immune responses to rhα-Gal A. Themost serious and most common adverse reactions reported withadministration of existing ERT are infusion reactions. Infusionreactions can include: tachycardia, hypertension, throat tightness,chest pain/tightness, dyspnea, fever, chills/rigors, abdominal pain,pruritus, urticaria, nausea, vomiting, lip or ear edema, and rash.

Most patients receiving Fabrazyme (agalsidase beta) develop IgGantibodies, some developing IgE or skin test reactivity specific to therecombinant enzyme. To ameliorate infusion reactions some patients arepretreated with acetaminophen and/or an antihistamine or steroids.Another strategy to control infusion reactions is to increase theinfusion time.

While different N-linked carbohydrates impart desirable characteristicsto the ERT, there are a limited number of glycosylation sites. Thus,design of ERT is not simply a case of adding desirable carbohydratesbecause choosing one characteristic or one type of carbohydrate may beat the expense of another characteristic. Accordingly, the developmentof a novel ERT presents many difficulties. The rhα-Gal A enzymesdescribed herein are the result of careful monitoring of the glycan mapduring ERT selection, expression, and purification to ensure minimizedoff-target clearance via the mannose and asialoglycoprotein receptorsand maximal productive high affinity targeting to the lysosome via theCIMPR.

Through diligent study and extensive experimentation the inventors havebalanced many protein characteristics to minimize non-productiveclearance of rhα-Gal A and efficiently target the rhα-Gal A tolysosomes. Among the characteristics that have been applied to therhα-Gal A described herein are: high production of protein,phosphorylation, terminal sialic acid capping of complex-type glycans,low content of neutral glycans, high enzyme activity, and stability inthe blood.

In at least one embodiment, the rhα-Gal A is expressed in Chinesehamster ovary (CHO) cells and comprises an increased content of N-glycanunits bearing one or more M6P residues when compared to a content ofN-glycan units bearing one or more M6P residues of Replagal (agalsidasealfa) or Fabrazyme (agalsidase beta). The rhα-Gal A as described hereinhas been shown to bind CIMPR with high affinity (K_(D)˜3 nM). Therhα-Gal A as described herein was characterized in vivo and shown tohave a longer apparent plasma half-life (t^(1/2)˜ 13.2 min) thanFabrazyme (agalsidase beta) (t^(1/2)˜7.8 min).

In at least one embodiment, the rhα-Gal A has both low levels of neutralglycans (e.g. 1.5-6%) and high levels of bis-phosphorylatedoligosaccharides (e.g. 7%-14%). In at least one other embodiment, therhα-Gal A has very high levels of mono-phosphorylated oligosaccharides(e.g. greater than 25%) and very high levels of bis-phosphorylatedoligosaccharides (e.g. greater than 12%). The rhα-Gal A may also havevery high levels of oligosaccharides containing sialic acid (e.g.greater than 50%). In these and other embodiments, the ERT has minimalnon-productive off-target clearance via the mannose andasialoglycoprotein receptors while maximizing productive high affinitytargeting to the lysosome via the CIMPR.

In at least one embodiment, the rhα-Gal A is a homodimer. In at leastone embodiment, the rhα-Gal A is initially expressed as having thefull-length 429 amino acid sequence of wild-type α-Gal A as set forth inSEQ ID NO: 1 and is associated with GenBank accession number NP_00160.1.The full-length rhα-Gal A undergoes intracellular processing thatremoves a portion of the amino acids, e.g. the first 31 amino acids.Accordingly, the rhα-Gal A that is produced and secreted by the hostcell can have a shorter amino acid sequence than the rhα-Gal A that isinitially expressed within the cell. In at least one embodiment, theshorter protein can have the amino acid sequence set forth in SEQ ID NO:2, which only differs from SEQ ID NO: 1 in that the first 31 amino acidscomprising the signal peptide have been removed, thus resulting in aprotein having 398 amino acids. In another embodiment each chain isapproximately 49 kDa before removal of the signal peptide andapproximately 45 kDa after removal of the signal peptide, not accountingfor the additional weight of glycans. Other variations in the number ofamino acids is also possible, such as having 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 or more deletions, substitutions and/orinsertions relative to the amino acid sequence described by SEQ ID NO: 1or SEQ ID NO: 2. In some embodiments, the rhα-Gal A product includes amixture of rhα-Gal A molecules having different amino acid lengths.

In at least one embodiment, the homodimer has 796 amino acids with amolecular weight of approximately 91 kDa, not accounting for theadditional weight of glycans.

SEQ ID NO: 1Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala LeuArg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala LeuAsp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp GluArg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys IleSer Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu GlyTrp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp MetAla Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln ArgPhe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys GlyLeu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala GlyPhe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe AlaAsp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp SerLeu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu AsnArg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr MetTrp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys AsnHis Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile LysSer Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp ValAla Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly AsnPhe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp AlaIle Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile SerPro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile AsnGln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp AsnPhe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val AlaMet Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile AlaVal Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe IleThr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp ThrSer Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu GlnLeu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu SEQ ID NO: 2Leu Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His TrpGlu Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser CysIle Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser GluGly Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys TrpMet Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro GlnArg Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser LysGly Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys AlaGly Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr PheAla Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys AspSer Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala LeuAsn Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu TyrMet Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr CysAsn His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser IleLys Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val AspVal Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile GlyAsn Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu TrpAla Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His IleSer Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala IleAsn Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly AspAsn Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala ValAla Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr IleAla Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys PheIle Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu TrpThr Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu LeuGln Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu SEQ ID NO: 3ATGCAGCTGAGGAATCCCGAGCTCCACCTGGGCTGTGCTCTGGCTCTGCGGTTCCTGGCCCTCGTGTCCTGGGACATCCCTGGCGCTAGGGCCCTCGATAACGGACTGGCCCGGACCCCCACAATGGGATGGCTCCACTGGGAAAGGTTCATGTGCAATCTGGACTGTCAGGAGGAACCCGACTCCTGCATCAGCGAAAAGCTCTTCATGGAGATGGCCGAGCTGATGGTGAGCGAGGGCTGGAAGGACGCCGGCTACGAGTATCTGTGCATCGATGACTGCTGGATGGCCCCTCAAAGGGACTCCGAAGGCAGGCTGCAGGCTGATCCCCAAAGGTTTCCCCACGGAATCCGGCAGCTCGCCAACTACGTGCATTCCAAGGGCCTCAAGCTCGGCATCTACGCCGACGTGGGCAACAAAACATGCGCCGGATTCCCCGGCAGCTTCGGCTACTACGACATCGACGCCCAGACATTCGCTGATTGGGGAGTGGACCTGCTGAAGTTCGACGGCTGTTACTGCGATTCCCTGGAAAACCTGGCCGACGGCTACAAACACATGTCCCTCGCCCTGAACCGGACAGGCAGGTCCATCGTGTACAGCTGCGAGTGGCCCCTGTACATGTGGCCTTTCCAGAAGCCCAACTACACAGAGATCAGGCAGTACTGCAACCACTGGAGGAACTTCGCTGACATCGACGACTCCTGGAAGAGCATCAAGAGCATCCTGGACTGGACCAGCTTCAACCAGGAGAGGATCGTGGACGTGGCTGGACCCGGAGGCTGGAACGACCCCGATATGCTGGTGATTGGCAACTTCGGACTGAGCTGGAACCAGCAGGTGACCCAGATGGCCCTGTGGGCCATTATGGCCGCTCCCCTGTTCATGTCCAACGACCTGAGGCACATCAGCCCCCAGGCCAAGGCTCTGCTGCAGGACAAGGATGTGATCGCCATCAACCAGGACCCCCTGGGCAAGCAGGGCTACCAGCTGAGGCAAGGAGATAACTTCGAGGTGTGGGAGAGGCCCCTGTCCGGACTGGCTTGGGCCGTGGCCATGATCAATCGGCAGGAGATCGGCGGACCCCGGTCCTACACCATTGCTGTGGCCAGCCTGGGAAAAGGAGTCGCCTGCAACCCCGCCTGCTTCATTACCCAGCTGCTCCCCGTGAAGCGGAAGCTGGGCTTCTATGAGTGGACCAGCAGGCTGAGGTCCCATATCAATCCTACCGGCACCGTCCTCCTCCAGCTCGAGAATACCATGCAGATGAGCCTCAAGGATCTGCTGTGA

In at least one embodiment, the rhα-Gal A undergoes post-translationaland/or chemical modifications at one or more amino acid residues in theprotein. For example, methionine and tryptophan residues can undergooxidation. As another example, the N-terminal glutamine can formpyro-glutamate. As another example, asparagine residues can undergodeamidation to aspartic acid. As yet another example, aspartic acidresidues can undergo isomerization to iso-aspartic acid. As yet anotherexample, unpaired cysteine residues in the protein can form disulfidebonds with free glutathione and/or cysteine. Accordingly, in someembodiments the enzyme is initially expressed as having an amino acidsequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2 or as encoded by SEQID NO: 3, and the enzyme undergoes one or more of thesepost-translational and/or chemical modifications. Such modifications arealso within the scope of the present disclosure.

In various embodiments, the rhα-Gal A is a variant that is at least 90%,95%, 98%, 99% or 99.5% identical to SEQ ID NO: 1 or SEQ ID NO: 2. Thesevariant α-galactosidase A amino acid sequences may contain deletions,substitutions and/or insertions relative to SEQ ID NO: 1 or SEQ ID NO:2, such as having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ormore deletions, substitutions and/or insertions relative to the aminoacid sequence described by SEQ ID NO: 1 or SEQ ID NO: 2.

Polynucleotide sequences encoding Gla and such variant human Glas arealso contemplated and may be used to recombinantly express rhα-Gal Aaccording to the invention.

The rhα-Gal A is preferably produced by CHO cells. DNA constructs whichmay express allelic variants of α-galactosidase A or other variantα-galactosidase amino acid sequences such as those that are at least90%, 95%, 98%, 99% or 99.5% identical to SEQ ID NO: 1 or SEQ ID NO: 2,may be constructed and expressed in CHO cells. These variantα-galactosidase A amino acid sequences may contain deletions,substitutions and/or insertions relative to SEQ ID NO: 1 or SEQ ID NO:2, such as having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ormore deletions, substitutions and/or insertions relative to the aminoacid sequence described by SEQ ID NO: 1 or SEQ ID NO: 2. The DNAconstructs can be at least 80%, 85%, 90%, 95%, 98%, 99% or 99.5%identical to SEQ ID NO: 3. Those of skill in the art can selectalternative vectors suitable for transforming CHO cells for productionof such variant α-galactosidase amino acid sequences.

Various alignment algorithms and/or programs may be used to calculatethe identity between two sequences, including FASTA, or BLAST which areavailable as a part of the GCG sequence analysis package (University ofWisconsin, Madison, Wis.), and can be used with, e.g., default setting.For example, polypeptides having at least 90%, 95%, 98% or 99% identityto specific polypeptides described herein and preferably exhibitingsubstantially the same functions, as well as polynucleotide encodingsuch polypeptides, are contemplated. Unless otherwise indicated asimilarity score will be based on use of BLOSUM62. When BLASTP is used,the percent similarity is based on the BLASTP positives score and thepercent sequence identity is based on the BLASTP identities score.BLASTP “Identities” shows the number and fraction of total residues inthe high scoring sequence pairs which are identical; and BLASTP“Positives” shows the number and fraction of residues for which thealignment scores have positive values and which are similar to eachother. Amino acid sequences having these degrees of identity orsimilarity or any intermediate degree of identity of similarity to theamino acid sequences disclosed herein are contemplated and encompassedby this disclosure. The polynucleotide sequences of similar polypeptidesare deduced using the genetic code and may be obtained by conventionalmeans, in particular by reverse translating its amino acid sequenceusing the genetic code.

Preferably, no more than 70, 65, 60, 55, 45, 40, 35, 30, 25, 20, 15, 10,or 5% of the total rhα-Gal A molecules lack an N-glycan unit bearing oneor more M6P residues or lacks a capacity to bind to the CIMPR.Alternatively, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,99%, <100% or more of the rhα-Gal A molecules comprise at least oneN-glycan unit bearing one or more M6P residues or has the capacity tobind to CIMPR.

The rhα-Gal A molecules may have 1, 2, 3 4, 5 or 6 M6P groups on theirglycans per subunit or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 M6Pgroups per homodimer. For example, only one N-glycan on a rhα-Gal Amolecule may bear M6P (mono-phosphorylated), a single N-glycan may beartwo M6P groups (bis-phosphorylated), or two different N-glycans on thesame rhα-Gal A molecule may each bear single M6P groups. The rhα-Gal Amolecules may also have N-glycans bearing no M6P groups. In anotherembodiment, on average the N-glycans contain greater than 2 mol/mol ofM6P and greater than 4 mol/mol sialic acid, such that the rhα-Gal Acomprises on average at least 2 moles of M6P residues per mole ofrhα-Gal A and at least 4 moles of sialic acid per mole of rhα-Gal Ahomodimer. On average at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 20, 25, 30 or 35% of the total glycans on the rhα-Gal A maybe in the form of a mono-M6P glycan, and on average, at least about 0.5,1, 1.5, 2.0, 2.5, 3.0, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20% of the total glycans on the rhα-Gal A are in the form of abis-M6P glycan and on average less than about 75, 70, 65, 60, 55, 50,45, 40, 35, 30 or 25% of the total glycans on the rhα-Gal A contains nophosphorylated glycan binding to CIMPR.

The rhα-Gal A may have an average content of N glycans carrying M6Pranging from 0.5 to 6.0 mol/mol rhα-Gal A homodimer or any intermediatevalue of subrange including 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5,5.0, 5.5, or 6.0 mol/mol rhα-Gal A homodimer. The rhα-Gal A can befractionated to provide rhα-Gal A preparations with different averagenumbers of mono-M6P-bearing or bis-M6P-bearing glycans thus permittingfurther customization of rhα-Gal A targeting to the lysosomes in targettissues by selecting a particular fraction or by selectively combiningdifferent fractions.

Up to 60% of the N-glycans on the rhα-Gal A may be fully sialylated, forexample, up to 10%, 20%, 30%, 40%, 50% or 60% of the N-glycans may befully sialylated. In some embodiments from 4 to 20% of the totalN-glycans are fully sialylated. In other embodiments no more than 5%,10%, 15%, 20%, 25% or 30% of N-glycans on the rhα-Gal A carry sialicacid and a terminal galactose residue (Gal). This range includes allintermediate values and subranges, for example, 7 to 30% of the totalN-glycans on the rhα-Gal A can carry sialic acid and terminal galactose.In yet other embodiments, no more than 5, 10, 15, 16, 17, 18, 19 or 20%of the N-glycans on the rhα-Gal A have a terminal galactose only and donot contain sialic acid. This range includes all intermediate values andsubranges, for example, from 8 to 19% of the total N-glycans on therhα-Gal A in the composition may have terminal galactose only and do notcontain sialic acid.

In other embodiments of the invention, 40, 45, 50, 55 or 60% of thetotal N glycans on the rhα-Gal A are complex-type N-glycans; or no morethan 1, 2, 3, 4, 5, 6, 7% of total N-glycans on the rhα-Gal A arehybrid-type N-glycans; no more than 5, 10, or 15% of the highmannose-type N-glycans on the rhα-Gal A are non-phosphorylated; at least5, 10, 15, 20, 25, 30 or 35% of the high mannose-type N-glycans on therhα-Gal A are mono-M6P phosphorylated; and/or at least 1, 2, 3, 4, 5, 6or 7% of the high mannose-type N-glycans on the rhα-Gal A are bis-M6Pphosphorylated. These values include all intermediate values andsubranges. The rhα-Gal A may meet one or more of the content rangesdescribed above.

In one or more embodiments, the rhα-Gal A will bear, on average, 2.0 to9.0 moles of sialic acid residues per mole of rhα-Gal A. This rangeincludes all intermediate values and subranges including 2.0, 2.5, 3.0,3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0 molresidues/mol rhα-Gal A homodimer. Without being bound by theory, it isbelieved that the presence of N-glycan units bearing sialic acidresidues may prevent non-productive clearance of the rhα-Gal A byasialoglycoprotein receptors.

In one or more embodiments, at least 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24 or 25% of the total N-glycans on the rhα-Gal Acontain a single sialic acid residue.

In one or more embodiments, at least 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% of the total N-glycanson the rhα-Gal A contain two sialic acid residues. These values includeall intermediate values and subranges.

In one or more embodiments, at least 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50% of the total N-glycanson the rhα-Gal A contain one or two sialic acid residues. These valuesinclude all intermediate values and subranges.

In one or more embodiments, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19 or 20% of the total N-glycans on the rhα-Gal Acontain three sialic acid residues. In one or more embodiments, lessthan 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10% of the totalN-glycans on the rhα-Gal A contain three sialic acid residues. Thesevalues include all intermediate values and subranges.

In one or more embodiments, at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5or 5% of the total N-glycans on the rhα-Gal A contain four sialic acidresidues. In one or more embodiments, less than 10, 9, 8, 7, 6, 5, 4.5,4, 3.5, 3, 2.5, 2, 1.5, 1 or 0.5% the total N-glycans on the rhα-Gal Acontain four sialic acid residues. These values include all intermediatevalues and subranges.

In one or more embodiments, less than 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0,5.5, 6.0, 6.5, 7.0, 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 20 or 25% of thetotal N-glycans on the rhα-Gal A are neutral glycans.

In one or more embodiments, the rhα-Gal A has M6P and/or sialic acidunits at certain N-glycosylation sites of the recombinant humanlysosomal protein. For example, there are three N-linked glycosylationsites on each of the two identical subunits of rhα-Gal A. There are fourpotential glycosylation sites corresponding to the positions of SEQ IDNO: 1: Asn-139, Asn-192, Asn-215 and Asn-408. Similarly, for maturepeptide of SEQ ID NO: 2, these potential glycosylation sites are atpositions: Asn-108, Asn-161, Asn-184 and Asn-377. Typically, only thefirst three sites are glycosylated. Other variants of rhα-Gal A can havesimilar glycosylation sites, depending on the location of asparagineresidues. Generally, sequences of ASN-X-SER or ASN-X-THR in the proteinamino acid sequence indicate potential glycosylation sites, with theexception that X cannot be HIS or PRO.

The inventors have found that rhα-Gal A having superior ability totarget CIMPR and cellular lysosomes as well as glycosylation patternsthat reduce its non-productive clearance in vivo can be produced CHOcells. These cells can express rhα-Gal A with significantly higherlevels of N-glycan units bearing one or more M6P residues thanconventional recombinant human α-galactosidase A products such asReplagal (agalsidase alfa) or Fabrazyme (agalsidase beta). The rhα-Gal Aproduced by these cells, for example, as exemplified in the Examplesbelow, has significantly more M6P and bis-M6P N-glycan residues and/orsignificantly more sialic acid N-glycan residues and/or significantlyless neutral N-glycans than conventional α-galactosidase A, such asReplagal (agalsidase alfa) or Fabrazyme (agalsidase beta). Without beingbound by theory, it is believed that this unique glycosylation allowsthe rhα-Gal A enzyme to be taken up more effectively into target cells,and therefore to be cleared from the circulation more efficiently thanother recombinant human α-galactosidase A molecules, such as forexample, Replagal (agalsidase alfa) or Fabrazyme (agalsidase beta),which has a much lower M6P and bis-M6P content and/or higher content ofneutral glycans. The rhα-Gal A as described herein has been shown toefficiently bind to CIMPR and be efficiently taken up by kidney andcardiac muscle and to have a glycosylation pattern that provides afavorable pharmacokinetic profile and reduces non-productive clearancein vivo.

It is also contemplated that the unique glycosylation of the rhα-Gal Aas described herein can contribute to a reduction of the immunogenicityof the rhα-Gal A compared to, for example, Replagal (agalsidase alfa) orFabrazyme (agalsidase beta). As will be appreciated by those skilled inthe art, glycosylation of proteins with conserved mammalian sugarsgenerally enhances product solubility and diminishes product aggregationand immunogenicity. Glycosylation indirectly alters proteinimmunogenicity by minimizing protein aggregation as well as by shieldingimmunogenic protein epitopes from the immune system (Guidance forIndustry—Immunogenicity Assessment for Therapeutic Protein Products, USDepartment of Health and Human Services, Food and Drug Administration,Center for Drug Evaluation and Research, Center for Biologics Evaluationand Research, August 2014). Therefore, in at least one embodiment,administration of the rhα-Gal A does not induce anti-drug antibodies. Inat least one embodiment, administration of the rhα-Gal A induces a lowerincidence of anti-drug antibodies in a subject than the level ofanti-drug antibodies induced by administration of Replagal (agalsidasealfa) or Fabrazyme (agalsidase beta).

Methods of Generating Cell Lines for Producing the rhα-Gal A

The rhα-Gal A having high content of mono-M6P, high content of bis-M6P,high content of sialic acid and/or low content neutral glycans can beproduced by transforming host cells with a DNA construct that encodesGla. Suitable host cells include mammalian cells, for example CHO cellssuch as CHO-DG44 cells, CHO-K1 cells, and CHO-DXB11 cells. While CHOcells have been previously used to make recombinant humanα-galactosidase A, it was not appreciated that transformed CHO cellscould be created and cultured in a way that would produce recombinantenzyme having a high content of mono-M6P, bis-M6P and sialicacid-containing glycans and/or have a low content of neutral glycans.

Suitable vectors for transfecting host cells can include DNA constructsthat are at least 80%, 85%, 90%, 95%, 98%, 99%. 99.5% or 100% identicalto SEQ ID NO: 3. Alternative DNA constructs may also be used to produceamino acid sequences that are at least 90%, 95%, 98%, 99%, 99.5% or 100%identical to SEQ ID NO: 1 or SEQ ID NO: 2. Exemplary vectors include,but are not limited to, plasmids. The vector(s) can include othersequences such as promoter sequences and/or sequences encoding selectiongenes. Promoter sequences include, but are not limited to, simian virus40 (SV40) and human cytomegalovirus (CMV) promoter genes. Selectiongenes include, but are not limited to, Zeocin resistance genes,ampicillin resistance genes, blasticidin S-resistance genes anddihydrofolate reductase genes. Other promoter sequences and selectiongenes are known in the art. Multiple vectors may also be used in atransfection, e.g. dual vector transfections.

Multiple transfections may be performed in host cells to provideseparate pools for evaluation. These pools may be evaluated for specificproperties, such as cell culture viability, rhα-Gal A expression and/orglycan content such as mono-M6P, bis-M6P, sialic acid and/or neutralglycan content. These properties can be evaluated throughout culturingsuch as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, etc. days aftertransfection.

Pools having desirable characteristics can be selected for generatingclones, and these clones can be further evaluated according to similarcriteria (e.g. cell culture viability, rhα-Gal A expression and/orglycan content). Selected clones can also be evaluated in otherconditions, on different scales (e.g. spin tube or bioreactor scales),or at different time points during culturing.

Methods of generating expression vectors, generating cell lines, andproducing recombinant cell lines are commonly used, but others may beused according to the knowledge of the skilled artisan. For example,such methods are described in: U.S. Patent App. Pub. No. US2016/0264953;International Publication No. WO 2014/137903; Brown et al., Trends inBiotechnology, vol. 25, Issue 9, September 2007, pp. 425-432; Tong etal., J. Biol. Chem., 264(14), 7970-7975 (1989); Matsuura et al,Glycobiology, 8(4): 329-339 (1998); and Dittermer et al., Biochem. J.,340:729-736 (1999), which are herein incorporated by reference in theirentireties.

Expression Vectors

Also provided herein are expression vectors containing a sequence (e.g.,cDNA) encoding recombinant human α-galactosidase-A proteins of thepresent invention. For example, the expression vector can include asequence (e.g., a cDNA) encoding recombinant human α-galactosidase-Aprotein that is at least 90% identical (e.g., at least 90%, 92%, 94%,96%, 98%, 99% or 100% identical) to SEQ ID NO: 1 or SEQ ID NO: 2. Inaddition to that sequence, the expression vectors can include a promoter(and optionally one or more enhancer sequences) operably linked to the5′ end of the sequence (e.g., cDNA) encoding the recombinant humanα-galactosidase-A protein. The expression vectors can include a sequenceencoding a peptide of human CD52 protein with a TTG start codon operablylinked to the 5′ end of the sequence (e.g., cDNA) encoding therecombinant protein and a sequence encoding a poly(A) recognition siteoperably linked to the 3′ end of the sequence (e.g., cDNA) encoding therecombinant protein. The expression vectors can include a sequence(e.g., cDNA) encoding a mammalian selection marker (e.g., a human or dogsynthetase protein) and a promoter sequence operably linked to the 5′end of the sequence (e.g., cDNA) encoding the mammalian selectionmarker, and optionally, a SV40 early intron sequence and poly(A) signalsequence that are both operably linked to the 3′ end of the sequence(e.g., cDNA) encoding the mammalian selection gene. Expression vectorscan include one or more (e.g., two or three) of: a prokaryotic promotersequence that is operably linked to the 5′ end of the sequence encodinga prokaryotic selection gene (e.g., an ampicillin resistance gene), aprokaryotic origin of replication sequence, and a eukaryotic origin ofreplication sequence.

Non-limiting examples of promoter sequences (and optionally one or moreenhancer sequence(s)) that can be operably linked to the sequence (e.g.,cDNA) encoding recombinant human α-galactosidase-A protein include:Simian Virus 40 (SV 40) early promoter, ribosomal protein 21 (rpS21)promoter, hamster S-actin promoter, cytomegalovirus (CMV) promoter(e.g., CMV immediate early promoter (see, e.g., Teschendorf et al.,Anticancer Res. 22:3325-3330, 2002), ubiquitin C (UBC) promoter,elongation factor 1-a (EF1A) promoter, phosphoenolpyruvate carboxykinase(PCK) promoter, IE2 promoter/enhancer region from mouse CMV (see, e.g.,Chatellard et al., Biotechnol. Bioeng. 96: 106-117, 2007), and chickenS-actin promoter. Additional non-limiting examples of human genepromoters that can be used in any of the expression vectors describedherein are described in the Mammalian Promoter Database (WistarInstitute website at mrpombdb.wister.upenn.edu). Additional examples ofmammalian promoter sequences that can be used in the expression vectorsare known in the art. One non-limiting example of a promoter and anenhancer that can be used in an expression plasmid is a chicken S-actinpromoter with a CMV enhancer (known in the art as a CAGG promoter). Theexpression vectors provided herein can include a rpS21 promoter, ahamster S-actin promoter, or a SV 40 early promoter sequence operablylinked to the 5′ end of the sequence (e.g., cDNA) encoding humanrecombinant α-galactosidase-A protein, a sequence encoding a peptide ofhuman CD52 protein with a TTG start codon operably linked to the 5′ endof the nucleic acid (e.g., cDNA) encoding human recombinantα-galactosidase-A protein (e.g., any of the nucleic acids encoding humanrecombinant α-galactosidase-A protein described herein), and a sequencecontaining a poly(A) recognition site operably linked to a 3′ end of thenucleic acid sequence encoding recombinant human α-galactosidase-Aprotein.

Non-limiting examples of poly(A) recognition site sequences are bovinegrowth hormone poly(A) recognition site. The structural features of ahuman poly(A) recognition site are described in Nunes et al., EMBO J29:1523-1536, 2010. Additional poly(A) recognitions sites are well-knownin the art.

In some examples, the expression vector includes a hamster S-actinpromoter and sequence encoding a peptide of human CD52 protein with aTTG start codon both operably linked to the 5′ end of the sequence(e.g., cDNA) encoding the recombinant human α-galactosidase-A protein,and a SV 40 early intron and poly(A) recognition sequence operablylinked to the 3′ end of the sequence (e.g., cDNA) encoding therecombinant human α-galactosidase-A protein.

Some expression vectors can include a sequence encoding a mammalianselection gene. Non-limiting examples of mammalian selection genesinclude: dihydrofolate reductase gene, hydromycin resistance genes,neomycin resistance genes, blasticidin resistance genes, zeocinresistance genes, glutamine synthetase genes, dihydrofolate resistancegenes, and hypoxanthine-guanine phosphoribosyltransferase genes.Examples of sequences encoding these mammalian selection genes are knownin the art. The 5′ end of the sequence encoding the mammalian selectiongene can be operably linked to a promoter (e.g., any of the exemplarypromoters described herein or known in the art).

Some expression vectors (e.g., any of the expression vector describedherein) can include a mammalian origin of replication sequence and/or aprokaryotic origin of replication sequence. Mammalian origin ofreplication sequences are known in the art (e.g., Todorovic et al.,Front. Biosci. 4:D859-D568, 1999; Aladjem, Front. Biosci. 9:2540-2547,2004; Hamlin, Bioessays 14:651-659, 1992). Prokaryotic origin ofreplication sequences are also known in the art (e.g., Marczynski etal., Curr. Opin. Genet. Dev. 3:775-782, 1993).

A non-limiting example of a vector is a plasmid. Non-limiting examplesof plasmids provided herein are shown in FIG. 3 .

An expression vector can be a viral vector. Non-limiting examples ofviral vectors include adenovirus vectors, herpes virus vectors,baculovirus vectors, and retroviral vectors. An expression vector canalso be a plasmid or a cosmid.

Host Cells

Also provided herein are host cells that include a sequence encodingrecombinant human α-galactosidase-A protein described herein. Thesequence can be operably linked to a promoter sequence (e.g., any of theexemplary promoter sequences described herein or any of the viral ormammalian promoter sequences known in the art). For example, thesequence encoding recombinant human α-galactosidase-A protein and thesequence of the promoter sequence operably linked to the 5′ end of thesequence encoding recombinant human α-galactosidase-A protein can beintegrated within a chromosome in the host cell. In other examples, thesequence encoding recombinant human α-galactosidase-A protein and thepromoter sequence that is operably linked to the 5′ end of the sequenceencoding recombinant human α-galactosidase-A protein are present in anexpression vector (e.g., any of the expression vectors described herein)within the host cell.

Methods for introducing nucleic acids (e.g., any of the nucleic acids orexpression vectors described herein) into a cell (e.g., a mammalian hostcell) are known in the art. For example, nucleic acid can be introducedinto a cell using lipofection, electroporation, calciumphosphate-mediated transfection, virus (e.g., retroviral) transduction,DEAE-dextran-mediated cell transfection, dendrimer-mediatedtransfection, sonoporation, optical transfection, impalefection,hydrodynamic delivery, magnetofection, or ballistic transfection.

A host cell can be any type of mammalian cell. For example, a host cellcan be a cell line, e.g., Chinese hamster ovary (CHO) cells (e.g., CHODG44 cells, CHO-K1s cells, C02.31 clonal cells,A14.13 clonal cells,C02.57 clonal cells, and F05.43 clonal cells), Sp2.0, myeloma cells(e.g., NS/0), B-cells, hybridoma cells, T-cells, human embryonic kidney(HEK) cells (e.g., HEK 293E and HEK 293F), African green monkey kidneyepithelial cells (Vero) cells, or Madin-Darby Canine (Cocker Spaniel)kidney epithelial cells (MDCK) cells. Additional mammalian cells thatcan be cultured using the methods described herein are known in the art.A host cell can be, e.g., an epithelial cell, an endothelial cell, alymphocyte, a kidney cell, a lung cell, a T-cell, a myeloma cell, or aB-cell. Some host cells can be grown in a suspension cell culture or inan adherent cell culture.

Methods of Generating a Mammalian Cell Line

Also provided herein are methods for generating a mammalian cell lineuseful for recombinant expression of a glycoprotein.

Also provided are methods of generating a mammalian cell line useful forrecombinant expression of a glycoprotein (e.g., any of the recombinantproteins described herein or known in the art) that include: (a)generating single-cell subclone cultures from the culture after thesequential culturing, and selecting a subclone culture that hasacceptable transfection efficiency, cell growth in serum-free culturemedium, and recombinant protein expression (e.g., selecting a subcloneculture that has the best transfection efficiency, cell growth, andrecombinant protein expression as compared to the other tested subclonecultures); (c) generating single-cell subclone cultures from theselected subclone culture in (a); and (d) selecting a single-cellsubclone culture generated in (b) that has acceptable transfectionefficiency, peak cell density (e.g., peak cell density in serum-freemedium), growth properties (e.g., growth in serum-free medium),volumetric productivity rate (VPR), and recombinant protein expression,where the selected subclone of (c) is useful for recombinant expressionof a glycoprotein (e.g., a subclone culture that has the besttransfection efficiency, peak cell density, growth properties, VPR, andrecombinant protein expression) as compared to the other tested subclonecultures).

Also provided herein are mammalian cells or mammalian cell linesproduced by any of the methods described herein. Non-limiting examplesof serum-dependent immortalized cell lines that can be used in any ofthe methods described herein include Chinese Hamster Ovary (CHO) cells,myeloma cells, B-cells, hybridoma cells, T-cells, human embryonic kidney(HEK) cells, African green monkey kidney epithelial cells (Vero) cells,and Madin-Darby Canine (Cocker Spaniel) kidney epithelial cells (MDCK)cells. Other serum-dependent immortalized cell lines that can be used inany of the methods described herein are known in the art. For example,the serum-dependent immortalized cell line can be an epithelial cellline, an endothelial cell line, a lymphocyte cell line, a kidney cellline, a lung cell line, a T-cell line, a myeloma cell line, or a B-cellline. In some examples, the serum-dependent immortalized cell line growsin suspension. In other examples, the serum-dependent immortalized cellline grows in adherent cell culture.

In some examples, the serum-dependent immortalized cell line does notendogenously express dihydrofolate reductase. The selected subclone(either the first or the second subclone selected in the methods) can begrown in suspension.

Methods for culturing an immortalized mammalian cell line are known inthe art. Methods for determining the transfection efficiency, cellgrowth in a serum-free culture medium, recombinant protein expression,peak cell density (e.g., peak viable cell density), cell growthproperties, volumetric productivity rate, and the glycosylation profileof a produced recombinant protein are well-known in the art. Forexample, transfection efficiency can be determined by detecting thelevel of expression of a reporter gene in an expression plasmidtransfected into the cell (e.g., the expression of such a reporter genecan be detected using fluorescence-assisted cell sorting). Recombinantprotein expression can, for example, be determined by detecting thelevels of the recombinant protein present in a tissue culture medium orwithin the cell using an antibody that specifically binds to therecombinant protein. Peak cell density and cell growth can be assessed,e.g., by measuring the cell density (e.g., viable cell density) overtime in a cell culture (e.g., using a hemocytometer or othercommercially available automated cell counters). The volumetricproductivity rate of a cell can be determined using methods known in theart by assessing the levels of recombinant protein present in a cellculture medium or within the cell over time. The glycosylation profileof a recombinant glycoprotein produced by a cell can be determined, forexample, using any of the methods described in the Examples section ofthe present specification (e.g., 2-anthilic acid (AA)-derivatization andHPLC with fluorescent detection).

As is well-known in the art, a mammalian cell line produced by themethods described herein can be stored at low temperature (e.g., below−20° C., below −30° C., below −40° C., below −50° C., below −60° C.,below −70° C., or below −80° C.) for future use. Methods for preparingstocks of a mammalian cell line for storage at low temperatures isdescribed, for example, in Hewitt, Methods Mal. Biol. 640:83-105, 2010,and Phelan, Curr. Protoc. Hum. Genet. Appendix 3:3G, 2006). In someexamples, the mammalian cell lines produced by the methods describedherein are not exposed to serum-containing and/or serum-containingculture medium (e.g., prior to storage and/or after storage). In someexamples, the mammalian cell lines produced by the methods describedherein are cultured only in animal-derived component (ADC)-free culturemedium. In some examples, the mammalian cell lines produced by themethods described herein are only cultured in serum-free, protein-free,chemically defined growth medium

Methods of Producing a Recombinant Glycoprotein

Also provided herein are methods of producing a recombinant glycoprotein(e.g., recombinant human α-galactosidase-A protein, or any recombinantglycoprotein known in the art). These methods include providing amammalian cell produced by any of the methods described herein,introducing into the cell a nucleic acid (e.g., an expression vector)that includes a sequence encoding a glycoprotein (e.g., recombinanthuman α-galactosidase-A protein and any other glycoprotein known in theart), culturing the cell in a serum-free culture medium under conditionssufficient to produce the glycoprotein, and harvesting the glycoproteinfrom the cell or the growth culture medium. Also provided arerecombinant glycoproteins (e.g., recombinant human α-galactosidase-Aprotein) produced by any of the methods described herein.

In some instances, the nucleic acid that includes a sequence encoding aglycoprotein is an expression vector (e.g., any of the expressionvectors described herein). In other examples, the nucleic acid thatincludes a sequence encoding a glycoprotein is integrated into achromosome of the mammalian cell.

In some examples, the culturing is performed using a bioreactor. Thebioreactor can have a volume of, e.g., between about 1 L to about 10,000L (e.g., between about 1 L to about 50 L, between about 50 L to about500 L, between about 500 L to about 1000 L, between 500 L to about 5000L, between about 500 L to about 10,000 L, between about 5000 L to about10,000 L, between about 1 Land about 10,000 L, between about IL andabout 8,000 L, between about 1 Land about 6,000 L, between about 1 Landabout 5,000 L, between about 100 Land about 5,000 L, between about 10Land about 100 L, between about 10 Land about 4,000 L, between about 10Land about 3,000 L, between about 10 L and about 2,000 L, or betweenabout 10 Land about 1,000 L). The amount of liquid culture mediumpresent in a bioreactor can be, e.g., between about between about 0.5 Lto about 5,000 L (e.g., between about 0.5 L to about 25 L, between about25 L to about 250 L, between about 250 L to about 500 L, between 250 Lto about 2500 L, between about 250 L to about 5,000 L, between about2500 L to about 5,000 L, between about 0.5 Land about 5,000 L, betweenabout 0.5 Land about 4,000 L, between about 0.5 Land about 3,000 L,between about 0.5 Land about 2,500 L, between about 50 Land about 2,500L, between about 5 L and about 50 L, between about 5 L and about 2,000L, between about 5 L and about 1,500 L, between about 5 L and about1,000 L, or between about 5 L and about 500 L). Culturing cells can beperformed, e.g., using a fed-batch bioreactor or a perfusion bioreactor.Culturing can be performed by fed-batch culturing or perfusion culturing(e.g., in a bioreactor).

The interior surface of any of the bioreactors described herein as isknown in the art, one or more ports for the sparging of O₂, CO₂, and N₂into the liquid culture medium, and a stir mechanism for agitating theliquid culture medium. The bioreactor can incubate the cell culture in acontrolled humidified atmosphere (e.g., at a humidity of greater than20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or a humidityof 100%). The bioreactor can also be equipped with a mechanical devicethat is capable of removing a volume of liquid culture medium from thebioreactor and optionally, a filter within the mechanical device thatremoves the cells from the liquid culture medium during the process oftransfer of the liquid culture medium out of the bioreactor (e.g., analternating tangential flow (ATF) or tangential flow filtration (TFF)system). Culturing can include exposing the liquid culture medium in thebioreactor to an atmosphere that includes at most or about 15% CO2(e.g., at most or about 14% CO2, 12% CO2, 10% CO2, 8% CO2, 6% CO2, 5%CO2, 4% CO2, 3% CO2, 2% CO2, or at most or about 1% CO2).

Culturing can be performed at a temperature of about 31° C. to about 40°C. Skilled practitioners will appreciate that the temperature can bechanged at specific time point(s) during the culturing, e.g., on anhourly or daily basis. For example, the temperature can be changed orshifted (e.g., increased or decreased) at about one day, two days, threedays, four days, five days, six days, seven days, eight days, nine days,ten days, eleven days, twelve days, fourteen days, fifteen days, sixteendays, seventeen days, eighteen days, nineteen days, or about twenty daysor more after the initial seeding of the bioreactor with the mammaliancell). For example, the temperature can be shifted upwards (e.g., achange of up to or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5,8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to orabout 20° C.). For example, the temperature can be shifted downwards(e.g., a change of greater than 0.05° C. or about 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0,5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or up to or about 20° C.). The culturing can be performedusing protein-free, serum-free, chemically-defined medium. Non-limitingexamples of such media are known in the art and are commerciallyavailable. Non-limiting examples of useful culture medium include, e.g.,CD CHO, Opti CHO, and Forti CHO (all available from Life Technologies;Grand Island, N.Y.), Hycell CHO medium (Thermo Fisher Scientific, Inc.;Waltham, Mass.), Ex-cell CD CHO Fusion medium (Sigma-Aldrich Co.; St.Louis, Mo.), and PowerCHO medium (Lonza Group, Ltd.; Basel,Switzerland).

The mammalian cell can be any of the mammalian cells described herein.For example, the mammalian cell can be a CHO cell. The mammalian cellcan be a cell that does not endogenously express dihydrofolate reductase(e.g., a CHO cell that does not endogenously express dihydrofolatereductase).

The recombinant glycoprotein can be an enzyme (e.g., humanα-galactosidase-A protein or any other glycoprotein known in the art).In some examples, the nucleic acid (e.g., expression vector) includes asequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 1. The nucleic acid(e.g., expression vector) can comprise a promoter sequence operablylinked to the 5′ end of the nucleic acid encoding the glycoprotein, asequence encoding a peptide of human CD52 protein with a TTG start codonoperably linked to the 5′ end of the nucleic acid encoding theglycoprotein, and a sequence encoding a poly(A) recognition siteoperably linked to the 3′ end of the nucleic acid encoding aglycoprotein. For example, the promoter sequence can be selected fromthe group consisting of: hamster rpS21 promoter, hamster S-actinpromoter, and SV 40 early promoter. The sequence encoding the poly(A)recognition site can be a SV 40 early poly(A) recognition sequence. Insome examples, the promoter sequence can be hamster S-actin promoter andthe poly(A) recognition sequence is a SV 40 early poly(A) recognitionsequence. In some examples, the nucleic acid further includes a sequenceencoding a human or dog glutamine synthetase (e.g., where the 5′ end ofthe nucleic acid encoding the human or dog glutamine synthetase isoperably linked to a SV 40 early promoter and the 3′ end of the nucleicacid encoding the human or dog glutamine synthetase is operably linkedto a SV 40 early intron and poly(A) signal sequence). In some examples,the nucleic acid further includes a sequence encoding a dihydrofolatereductase (DHFR) (e.g., human or mouse DHFR) (e.g., where the 5′ end ofthe nucleic acid encoding the dihydrofolate reductase is operably linkedto a SV 40 early promoter and the 3′ end of the nucleic acid encodingthe dihydrofolate reductase is operably linked to a SV 40 early intronand poly(A) signal sequence).

The culturing can be performed using a perfusion bioreactor. Perfusionculturing is well-known in the art and includes removing from abioreactor a first volume of a first liquid culture medium (e.g., thatincludes any concentration of mammalian cells, e.g., a first volume of afirst liquid culture medium that is substantially free of cells), andadding to the first liquid culture medium a second volume of a secondliquid culture medium. Removal and adding can be performedsimultaneously or sequentially, or a combination of the two. Further,removal and adding can be performed continuously (e.g., at a rate thatremoves and replaces a volume of between 0.1% to 800% (e.g., between 1%and 700%, between 1% and 600%, between 1% and 500%, between 1% and 400%,between 1% and 350%, between 1% and 300%, between 1% and 250%, between1% and 100%, between 100% and 200%, between 5% and 150%, between 10% and50%, between 15% and 40%, between 8% and 80%, and between 4% and 30%) ofthe volume of the bioreactor or the first liquid culture medium volumeover any given time period (e.g., over a 24-hour period, over anincremental time period of about 1 hour to about 24 hours, or over anincremental time period of greater than 24 hours)) or periodically(e.g., once every third day, once every other day, once a day, twice aday, three times a day, four times a day, or five times a day), or anycombination thereof. Where performed periodically, the volume that isremoved or replaced (e.g., within about a 24-hour period, within anincremental time period of about 1 hour to about 24 hours, or within anincremental time period of greater than 24 hours) can be, e.g., between0.1% to 800% (e.g., between 1% and 700%, between 1% and 600%, between 1%and 500%, between 1% and 400%, between 1% and 300%, between 1% and 200%,between 1% and 100%, between 100% and 200%, between 5% and 150%, between10% and 50%, between 15% and 40%, between 8% and 80%, and between 4% and30%) of the volume of the bioreactor or the first liquid culture mediumvolume. The first volume of the first liquid culture medium removed andthe second volume of the second liquid culture medium added can in someinstances be held approximately the same over each 24-hour period (or,alternatively, an incremental time period of about 1 hour to about 24hours or an incremental time period of greater than 24 hours) over theentire or part of the culturing period. As is known in the art, the rateat which the first volume of the first liquid culture medium is removed(volume/unit of time) and the rate at which the second volume of thesecond liquid culture medium is added (volume/unit of time) can bevaried. The rate at which the first volume of the first liquid culturemedium is removed (volume/unit of time) and the rate at which the secondvolume of the second liquid culture medium is added (volume/unit oftime) can be about the same or can be different.

Alternatively, the volume removed and added can change (e.g., graduallyincrease) over each 24-hour period (or alternatively, an incrementaltime period of between 1 hour and about 24 hours or an incremental timeperiod of greater than 24 hours) during the culturing period. Forexample the volume of the first liquid culture medium removed and thevolume of the second liquid culture medium added within each 24-hourperiod (or alternatively, an incremental time period of between about 1hour and above 24 hours or an incremental time period of greater than 24hours) over the culturing period can be increased (e.g., gradually orthrough staggered increments) over the culturing period from a volumethat is between 0.5% to about 20% of the bioreactor volume or the firstliquid culture medium volume to about 25% to about 150% of thebioreactor volume or the first liquid culture medium volume.

Skilled practitioners will appreciate that the first liquid culturemedium and the second liquid culture medium can be the same type ofmedia (e.g., serum-free or serum-free, protein-free chemically-definedmedium). In other instances, the first liquid culture medium and thesecond liquid culture medium can be different.

The first volume of the first liquid culture medium can be removed,e.g., using a mechanical system and/or by seeping or gravity flow of thevolume through a sterile membrane with a molecular weight cut-off thatexcludes mammalian cells present in the volume. The second volume of thesecond liquid culture medium can be added to the first liquid culturemedium in an automated fashion, e.g., by perfusion pump. In someinstances, removing the first volume of the first liquid culture medium(e.g., a first volume of the first liquid culture medium that issubstantially free of mammalian cells) and adding to the first liquidculture medium a second volume of the second liquid culture medium doesnot occur within at least 1 hour (e.g., within 2 hours, within 3 hours,within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8hours, within 9 hours, within 10 hours, within 12 hours, within 14hours, within 16 hours, within 18 hours, within 24 hours, within 36hours, within 48 hours, within 72 hours, within 96 hours, or after 96hours) of the seeding of the bioreactor with a mammalian cell.

Alternatively or in addition, culturing can be performed using afed-batch bioreactor. Such culturing is known in the art and includes,over the majority of the culturing period, addition (e.g., periodic orcontinuous addition) to the first liquid culture medium of a secondvolume of a second liquid culture medium. Adding of the second liquidculture medium can be performed continuously (e.g., at a rate that addsa volume of between 0.1% to 300% (e.g., between 1% and 250%, between 1%and 100%, between 100% and 200%, between 5% and 150%, between 10% and50%, between 15% and 40%, between 8% and 80%, and between 4% and 30%) ofthe volume of the bioreactor or the first liquid culture medium volumeover any given time period (e.g., over a 24-hour period, over anincremental time period of about 1 hour to about 24 hours, or over anincremental time period of greater than 24 hours)) or periodically(e.g., once every third day, once every other day, once a day, twice aday, three times a day, four times a day, or five times a day), or anycombination thereof. Where performed periodically, the volume that isadded (e.g., within about a 24-hour period, within an incremental timeperiod of about 1 hour to about 24 hours, or within an incremental timeperiod of greater than 24 hours) can be, e.g., between 0.1% to 300%(e.g., between 1% and 200%, between 1% and 100%, between 100% and 200%,between 5% and 150%, between 10% and 50%, between 15% and 40%, between8% and 80%, and between 4% and 30%) of the volume of the bioreactor orthe first liquid culture medium volume. The second volume of the secondliquid culture medium added can in some instances be held approximatelythe same over each 24-hour period (or, alternatively, an incrementaltime period of about 1 hour to about 24 hours or an incremental timeperiod of greater than 24 hours) over the entire or part of theculturing period. As is known in the art, the rate at which the secondvolume of the second liquid culture medium is added (volume/unit oftime) can be varied over the entire or part of the culturing period. Forexample, the volume of the second liquid culture medium added can change(e.g., gradually increase) over each 24-hour period (or alternatively,an incremental time period of between 1 hour and about 24 hours or anincremental time period of greater than 24 hours) during the culturingperiod. For example the volume of the second liquid culture medium addedwithin each 24-hour period (or alternatively, an incremental time periodof between about 1 hour and above 24 hours or an incremental time periodof greater than 24 hours) over the culturing period can be increased(e.g., gradually or through staggered increments) over the culturingperiod from a volume that is between 0.5% to about 20% of the bioreactorvolume or the first liquid culture medium volume to about 25% to about150% of the bioreactor volume or the first liquid culture medium volume.The rate at which the second volume of the second liquid culture mediumis added (volume/unit of time) can be about the same over the entire orpart of the culturing period.

Skilled practitioners will appreciate that the first liquid culturemedium and the second liquid culture medium can be the same type ofmedia (e.g., a protein-free culture medium or a serum-free, protein-freechemically-defined medium). In other instances, the first liquid culturemedium can be of a type that is different from the second liquid culturemedium. The volume of the second liquid culture medium can be added tothe first liquid culture medium in an automated fashion, e.g., byperfusion pump.

In some instances, adding to the first liquid culture medium a secondvolume of the second liquid culture medium does not occur until at least1 hour after but no more than 7 days after the seeding of the bioreactorwith a mammalian cell (e.g., until at least 2 hours, within 3 hours,within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8hours, within 9 hours, within 10 hours, within 12 hours, within 14hours, within 16 hours, within 18 hours, within 24 hours, within 36hours, within 48 hours, within 72 hours, within 96 hours, or after 96hours after, but not more than 7 da7s after the seeding of thebioreactor with a mammalian cell). The cell culture medium in fed-batchcultures is typically harvested at the end of culture period, however,the cell culture medium in fed-batch cultures can also be harvested atone or more time points during the culturing period.

Skilled practitioners will appreciate that any of the various parametersfor culturing (e.g., bioreactor, volumes, rates or frequencies ofreplacing culture volumes, agitation, temperatures, culture media,and/or CO2 concentrations) recited herein can be used in any combinationin performing these methods.

An additional step of isolating the recombinant glycoprotein can beperformed. As is well-known in the art, such methods differ according tothe physical properties and activities of the glycoprotein. For example,parameters such as the binding specificity of the glycoprotein (e.g.,substrate or antigen-binding activity), net charge, and/or size shouldbe considered when designing the steps for isolating a recombinantglycoprotein (e.g., from culture medium or from a cell). One or more ofany of the following methods can be used to isolate a recombinantglycoprotein (e.g., a recombinant glycoprotein produced using any of themethods described herein): affinity column chromatography, ionic (e.g.,cationic or anionic) exchange column chromatography, size exclusioncolumn chromatography, reverse-phase column chromatography, filtration,and precipitation. Non-limiting methods for isolating recombinant humanα-galactosidase-A protein are described in the Examples.

The methods described herein can further include formulating theisolated recombinant glycoprotein into a pharmaceutically acceptableexcipient or buffer (e.g., for administration to a subject). Thesemethods can further include sterile filtering, viral inactivation, UVirradiation, and/or lyophilization, or any combination thereof.

Production, Capturing and Purification of Rhα-Gal A

A typical cell manufacturing process begins with the revival ofcryopreserved cells from a master cell bank. Cells are thawed in smallvolume vials and progressively introduced to larger volume culturevessels to achieve inoculation of a seed bioreactor. The expanded cellpopulation in the seed bioreactor is then introduced into the productionbioreactor where the therapeutic protein will be expressed.

There are many types of reactor with the choice of reactor determinedempirically based on factors such as optimal cell growth, viability,protein yield, and maintenance of critical quality attributes of thetherapeutic protein. Batch, fed-batch, batch refeed or intermittentharvest, and perfusion are currently the most frequently usedbioreactors.

Batch culture is a closed system where culture media and cells are addedat the start of production and then cultured without any other additionsuntil the production run is terminated. The growth of cells progressesin four stages: lag phase, exponential growth phase, stationary phase,and death phase.

During a fed-batch cell culture a feed solution is added when cellculture components have been depleted. The feed solution contains aminoacids, vitamins, and trace elements in a concentrated form. Thus, withthe fed-batch culture the culture volume increases with each sequentialaddition of a feed solution.

A batch refeed or intermittent harvest culture removes a portion of thecell culture (including the cells) when culture nutrients have beendepleted and the cells are entering stationary phase. This method ofharvest is useful for labile proteins because the harvest can beperformed frequently such as every 2-4 days.

Cells in culture have a high energy need during exponential phase andrapidly convert glucose to lactate. High lactate levels are detrimentalto cell growth and productivity. Thus, lactate must be closelycontrolled to ensure product quality. Substitution of galactose ormannose for glucose may alleviate lactate build up because these sugarsare more slowly metabolized. Other culture parameters that affect growthkinetics, cell viability, and protein production are pH, temperature,and dissolved oxygen.

After the initial cell expansion, in perfusion cell culture the cellsare kept at a steady state by the addition of fresh culture media and aconcomitant removal of spent culture media that contains the proteinproduct. Higher cell densities are possible with perfusion culture thanwith batch culture because culture media is continuously replenished.Cells are retained in the culture while spent media containing productis removed. Periodically some media containing cells is removed to keepcell density within a productive range. Perfusion culture is alsocompatible with continuous processing.

Biologic drug production generates not one uniform protein but an arrayof different isoforms that reflect the complexity of biologicalprocesses. Protein variation can arise including glycosylation,phosphorylation, sulfation, amidation, oxidation, adduct formation,pyroglutamate formation and isomerization. Amino acid variation canarise from genetic mutations, amino acid misincorporation, clipping andN- and C-terminal heterogeneity. Structural variation can includemisfolding, aggregation, and disulfide scrambling.

Impurities introduced during manufacturing can also affect proteinstability, toxicity, and efficacy. Impurities of this type include hostcell proteins, DNA, media elements and viral components.

Proteins designed to be secreted from the cell have their signalsequences cleaved by signal peptidase upon translocation through theendoplasmic reticulum. Protein variants can be generated bydiscrepancies in the cleavage of the signal sequence. N-terminalglutamic acid or glutamine can cyclize to form pyroglutamate. This leadsto blocked proteins that may or may not have an effect on theirbiological function.

The two main types of glycosylation displayed on proteins is N-linkedand 0-linked glycosylation. As mentioned above, α-galactosidase A hasfour potential N-linked glycosylation sites on the monomer subunit,three of which are typically glycosylated. Glycosylation occurs at theconsensus sequence ASN-X-SER/THR where X is any amino acid other thanhistidine or proline. The cell line has a large influence of the type ofsugars incorporated at the glycosylation site as well as the cultureconditions and downstream processing of the protein.

Cysteine is a relatively reactive amino acid and an unpaired cysteinecan problematically react with other sulfhydryl groups with in cell,culture media, or cause intra- or inter-subunit bond formation.

Proteins may also be degraded or have poor product characteristics as aresult of the uncontrolled action of proteases, glycosidases, andphosphatases.

In various embodiments, a protein capturing system including one or moreanion exchange (AEX) columns is used for the direct product capture ofrhα-Gal A, particularly rhα-Gal A having a high M6P content, a highsialic acid content, and a low content of neutral glycans. While notwishing to be bound by any particular theory, it is believed that usingAEX chromatography to capture the rhα-Gal A from the filtered mediaensures that the captured recombinant protein product has a higher M6Pcontent and a higher sialic acid content, due to the more negativecharge of the recombinant protein having one or more M6P groups and/orsialic acid groups. As a result, non-phosphorylated recombinant proteinand host cell impurities do not bind the column resin as well as thehighly phosphorylated recombinant protein, and the non-phosphorylatedrecombinant protein and host cell impurities passes through the column.Accordingly, the AEX chromatography can be used to enrich the M6Pcontent of the protein product (i.e. select for protein molecules havingmore M6P) due to the high affinity of the M6P-containing proteins forthe AEX resin. Similarly, the AEX chromatography can be used to enrichthe sialic acid content of the protein product (i.e. select for proteinmolecules having more sialic acid) due to the affinity of the sialicacid-containing proteins for the AEX resin.

Furthermore, while not wishing to be bound by any particular theory, itis also believed that having a direct product capture of recombinantprotein using AEX chromatography ensures that the recombinant proteinshaving high M6P content are removed from the media containing proteasesand other enzymes that can degrade the protein and/or dephosphorylatethe protein. As a result, the high quality product is preserved.

Strong binding to an AEX column confirms three product characteristicsthat are desirable: phosphorylated enzyme, adequately sialylated enzymeand enzymes with low content of neutral glycans. At physiological pH,phosphorylation and sialylation impart a negative charge and contributeto binding to AEX columns. Enzyme that possesses mono-M6P and/or bis-M6Pwill bind the CIMPR which in turn targets the enzyme to the lysosome(site of enzyme action). Recombinant enzyme that has been adequatelysialylated also exhibits a greater half-life in the blood. This isbecause asialoglycoprotein receptors (located largely on liver cells)bind exposed galactose residues on proteins and remove the protein fromcirculation. Sialic acid on the terminal sugar residue blocks thismechanism of removal based on galactose.

Purification on AEX columns selects for phosphorylated/sialylated enzymewith low content of neutral glycans, if molecules with suchcharacteristics are present in the cell culture supernatant. The bindingto the CIMPR can be assessed by binding the enzyme preparation toimmobilized CIMPR followed by elution with increasing concentrations ofM6P.

Thus, one or more embodiments of the present invention relate to arecombinant α-Gal A with specific level of surface mono-M6P and/orbis-M6P and/or sialic acid and/or low content of neutral glycans, suchas rhα-Gal A produced by the methods described herein. The rhα-Gal Aproduced by such methods can have any of the characteristics describedherein.

Also, the chromatography techniques described herein can be utilized toenrich the mono-M6P and/or bis-M6P and/or sialic acid content of theenzyme molecules, relative to the enzyme molecules that are initiallypresent in the cell supernatant. As described above, the AEX column canbe used to select for enzyme molecules that have high content ofphosphorylation, high content of sialic acid, and low content of neutralglycans. Other columns such as CIMPR columns can be used to selectivelybind enzyme with high content of mono-M6P and bis-M6P. Thus, even if thehost cells initially express enzyme molecules with glycan contentsimilar to conventional recombinant human α-galactosidase A productssuch as Replagal (agalsidase alfa) or Fabrazyme (agalsidase beta), usingAEX and CIMPR columns during product capture and purification can beused to enrich the mono-M6P and/or bis-M6P and/or sialic acid contentand/or to lower the neutral glycan content of the enzyme molecules inthe final recombinant protein product relative to the enzyme moleculesthat are initially present in the cell supernatant.

Pharmacological Chaperones

Migalastat hydrochloride (HCl) is a low molecular weight iminosugar thatacts as a pharmacological chaperone for α-Gal A. It is an analog of theterminal galactose group that is cleaved from the substrateglobotriaosylceramide (GL-3). Pharmacological chaperones are designed tobind and stabilize the intended protein target to help restore properintracellular trafficking before dissociating from the protein, therebyallowing it to function as intended in the lysosome. As such, migalastatHCl is a potent, reversible, and competitive inhibitor of α-Gal A. Itbinds to mutant and wild-type forms of α-Gal A, stabilizes the enzyme,thus enabling the correctly folded protein to pass through theendoplasmic reticulum for proper trafficking to the lysosomes.

The relatively neutral pH of blood presents a hostile environment forlysosomally adapted enzymes used in enzyme replacement therapies becausethe ERTs are more stable in the acidic environment of the lysosome.Consequently, a proportion of the administered ERT denatures whichbecomes an antigen priming immune responses directed against theprotein. At the very least, the denatured enzyme is cleared from thebloodstream via a non-efficacious pathway. Migalastat binds to wild-typeα-Gal A ERT in the bloodstream, resulting in a stabilized enzyme withgreater activity and greater uptake into the lysosome via the CIMPR. Thelow pH and high concentration of accumulated substrate in the lysosomesfavor dissociation of migalastat HCl, allowing α-Gal A to bind andbreakdown GL-3.

In one or more embodiments, the rhα-Gal A is co-formulated with thepharmacological chaperone such as migalastat.

In one or more embodiments, the co-formulation composition comprisesα-Gal A at a concentration of between about 0.05 and about 100 μM, orbetween about 0.1 and about 75 μM, or between about 0.2 and about 50 μM,or between about 0.3 and about 40 μM, or between about 0.4 and about 30μM, or between about 0.5 and about 20 μM, or between about 0.6 and about15 μM, or between about 0.7 and about 10 μM, or between about 0.8 andabout 9 μM, or between about 0.9 and about 8 μM, or between about 1 andabout 7 μM, or between about 2 and about 6 μM, or between about 3 andabout 5 μM. Concentrations and ranges intermediate to the above recitedconcentrations are also intended to be part of this application.

In one or more embodiments, the co-formulation composition comprisesα-Gal A at a concentration of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1, 1.1, 1.2, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5,or 15 μM.

In one or more embodiments, the co-formulation composition comprisesα-Gal A at a concentration of between about 0.0025 and about 5 mg/ml, orbetween about 0.005 and about 4.5 mg/ml, or between about 0.025 andabout 4 mg/ml, or between about 0.05 and about 3.5 mg/ml, or betweenabout 0.25 and about 3 mg/ml, or between about 0.5 and about 2.5 mg/ml,or between about 0.75 and about 2 mg/ml, or between about 1 and about1.5 mg/ml. Concentrations and ranges intermediate to the above recitedconcentrations are also intended to be part of this application.

In one or more embodiments, the co-formulation composition comprisesmigalastat or salt thereof at a concentration of between about 10 andabout 25,000 μM, or between about 50 and about 20,000 μM, or betweenabout 100 and about 15,000 μM, or between about 150 and about 10,000 μM,or between about 200 and about 5,000 μM, or between about 250 and about1,500 μM, or between about 300 and about 1,000 μM, or between about 350and about 550 μM, or between about 400 and about 500 μM. Concentrationsand ranges intermediate to the above recited concentrations are alsointended to be part of this application.

In one or more embodiments, the co-formulation composition comprisesmigalastat or salt thereof at a concentration of between about 0.002 andabout 5 mg/ml, or between about 0.005 and about 4.5 mg/ml, or betweenabout 0.02 and about 4 mg/ml, or between about 0.05 and about 3.5 mg/ml,or between about 0.2 and about 3 mg/ml, or between about 0.5, and about2.5 mg/ml, or between about 1 and about 2 mg/ml. Concentrations andranges intermediate to the above recited concentrations are alsointended to be part of this application.

In one or more embodiments, the co-formulation composition comprisesmigalastat or salt thereof at a concentration of about 50; 100; 150;200; 250; 300; 350; 400; 450; 500; 550; 600; 650; 700; 750; 800; 850;900; 950; 1,000; 2,000; 3,000; 4,000; 5,000; 6,000; 7,000; 8,000; 9,000;10,000; 11,000; 12,000; 13,000; 14,000; 15,000; 16,000; 17,000; 18,000;19,000 or 20,000 μM.

In one or more embodiments, the α-Gal A enzyme and migalastat (or saltthereof) are combined to create a co-formulation with a molar ratio ofmigalastat to α-Gal A enzyme of between about 10:1 and about 20,000:1;or between about 20:1 and about 15,000:1; or between about 50:1 andabout 10,000:1; or between about 100:1 and about 5,000:1; or betweenabout 150:1 and about 1,000:1; or between about 200:1 and about 500:1;or between about 250:1 and about 450:1; or between about 300:1 and about400:1. Molar ratios and ranges intermediate to the above recited molarratios are also intended to be part of this application.

Methods of Treatment

The present invention provides a method of treating Fabry disease in apatient in need thereof, the method including administering rhα-Gal A asdescribed herein to a patient or contacting cells with rhα-Gal A asdescribed herein. In one or more embodiments, the rhα-Gal A is expressedin CHO cells and comprises an increased content of N-glycan unitsbearing one or two M6P residues when compared to a content of N-glycanunits bearing one or two M6P residues of Replagal (agalsidase alfa) orFabrazyme (agalsidase beta) and/or a substantially lower amount ofneutral glycans. The cell line producing the rhα-Gal A, the conditionsto express and the methods to purify have all been carefully picked,evaluated and scrutinized to maximize the potential benefits of having aglycosylated protein that minimizes non-productive targeting andmaximizes productive uptake into target cells.

The present invention also provides a method of treating Fabry diseasein a patient in need thereof, the method including administering apharmacological chaperone (e.g. migalastat or salt thereof), or apharmaceutically acceptable salt thereof, to the patient in combinationwith rhα-Gal A as described herein. In one or more embodiments, therhα-Gal A is expressed in CHO cells and comprises an increased contentof N-glycan units bearing one or two M6P residues when compared to acontent of N-glycan units bearing one or two M6P residues of Replagal(agalsidase alfa) or Fabrazyme (agalsidase beta) and a substantiallylower amount of neutral glycans. In another aspect, the presentinvention provides the use of a pharmacological chaperone (e.g.migalastat or salt thereof) and the rhα-Gal A in combination for thetreatment of Fabry disease in a patient in need thereof.

The present invention also provides a method of reducing the level ofGL-3 in an organ of a patient in need, the method comprisingadministering to the patient a composition comprising a therapeuticallyeffective amount of rhα-Gal A, optionally in combination with apharmacological chaperone. In one or more embodiments, the organ isheart, kidney or skin.

The present invention also provides a method of treating Fabry disease,the method comprising contacting a mammalian cell with an effectiveamount of rhα-Gal A, optionally in combination with a pharmacologicalchaperone, wherein contacting the cell with the rhα-Gal A provides agreater reduction in GL-3 than contacting with Fabrazyme (agalsidasebeta). In one or more embodiments, the contacting is administering aneffective to a subject an effective amount of the rhα-Gal A.

The present invention also provides a method of treating Fabry disease,the method comprising contacting a mammalian cell with an effectiveamount of rhα-Gal A, optionally in combination with a pharmacologicalchaperone, wherein contacting the cell with the rhα-Gal A provides agreater reduction in plasma lyso-Gb3 than contacting with Fabrazyme(agalsidase beta). In one or more embodiments, the contacting isadministering an effective to a subject an effective amount of therhα-Gal A.

The present invention also provides a method of treating Fabry disease,the method comprising administering a patient in an effective amount ofrhα-Gal A, optionally in combination with a pharmacological chaperone,wherein contacting the cell with the rhα-Gal A provides a greaterreduction in one or more substrates than contacting with Fabrazyme(agalsidase beta). In one or more embodiments, the contacting isadministering an effective to a subject an effective amount of therhα-Gal A. In one or more embodiments, the substrate comprises GL-3and/or plasma lyso-Gb3

The present invention also provides a method of enhancing the activitylevel of α-galactosidase-A protein in a lysosome in a mammalian cell,the method comprising contacting the mammalian cell with rhα-Gal A,optionally in combination with a pharmacological chaperone. In one ormore embodiments, the contacting is performed by administration of therhα-Gal A and optionally a pharmacological chaperone. In one or moreembodiments, the cell is in vitro.

In any of the above methods, the cell can be in a subject. In one ormore embodiments, the cell is located in the subject's heart. In one ormore embodiments, the cell is located in the subject's kidney. In one ormore embodiments, the cell is located in the subject's skin. In at leastone embodiment, the pharmacological chaperone (e.g. migalastat or saltthereof) is administered orally. In at least one embodiment, themigalastat or salt thereof is administered at an oral dose of about 100mg to about 400 mg, or at an oral dose of about 100 mg, about 150 mg,about 200 mg, about 250 mg or about 300 mg. In at least one embodiment,the migalastat or salt thereof is administered at an oral dose of about233 mg to about 400 mg. In at least one embodiment, the migalastat orsalt thereof is administered at an oral dose of about 250 to about 270mg, or at an oral dose of about 250 mg, about 255 mg, about 260 mg,about 265 mg or about 270 mg. In at least one embodiment, the migalastator salt thereof is administered as an oral dose of about 260 mg.

In at least one embodiment, the pharmacological chaperone (e.g.migalastat or salt thereof) is administered systemically, such asintravenously. In at least one embodiment, the migalastat or saltthereof is administered at an intravenous dose of about 100 mg to about400 mg, or at an intravenous dose of about 100 mg, about 123 mg, about150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg or about400 mg. In at least one embodiment, the migalastat or salt thereof isadministered at an intravenous dose of about 233 mg to about 400 mg. Inat least one embodiment, the migalastat or salt thereof is administeredat an intravenous dose of about 250 to about 270 mg, or at anintravenous dose of about 250 mg, about 255 mg, about 260 mg, about 265mg or about 270 mg. In at least one embodiment, the migalastat or saltthereof is administered as an intravenous dose of about 260 mg. In atleast one embodiment, the migalastat or salt thereof is administered atan intravenous dose of about 0.3 mg/kg to about 300 mg/kg, or at anintravenous dose of about 1 mg/kg to about 100 mg/kg. In at least oneembodiment, the migalastat or salt thereof is administered at anintravenous dose of about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about9 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg,about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg or about 100mg/kg.

In at least one embodiment, the pharmacological chaperone (e.g.migalastat or salt thereof) is administered subcutaneously. In at leastone embodiment, the migalastat or salt thereof is administered at asubcutaneous dose of about 100 mg to about 400 mg, or at a subcutaneousdose of about 100 mg, about 123 mg, about 150 mg, about 200 mg, about250 mg, about 300 mg, about 350 mg or about 400 mg. In at least oneembodiment, the migalastat or salt thereof is administered at asubcutaneous dose of about 233 mg to about 400 mg. In at least oneembodiment, the migalastat or salt thereof is administered at asubcutaneous dose of about 250 to about 270 mg, or at a subcutaneousdose of about 250 mg, about 255 mg, about 260 mg, about 265 mg or about270 mg. In at least one embodiment, the migalastat or salt thereof isadministered as a subcutaneous dose of about 260 mg.

It will be understood by those skilled in the art that an oral dose ofmigalastat in the range of about 100 mg to 400 mg or any smaller rangetherewithin can be suitable for an adult patient with an average bodyweight of about 70 kg. For patients having a significantly lower bodyweight than about 70 kg, including but not limited to infants, childrenor underweight adults, a smaller dose may be considered suitable by aphysician. Therefore, in at least one embodiment, the migalastat or saltthereof is administered as an oral dose of from about 25 mg to about 200mg, or as an oral dose of about 50 mg, about 75 mg, about 100 mg, 125mg, about 150 mg, about 175 mg or about 200 mg. In at least oneembodiment, the migalastat or salt thereof is administered as an oraldose of from about 65 mg to about 195 mg, or as an oral dose of about 65mg, about 130 mg or about 195 mg.

The rhα-Gal A can be formulated in accordance with the routineprocedures as a pharmaceutical composition adapted for administration tohuman beings. For example, in a preferred embodiment, a composition forintravenous administration is a solution in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic to ease pain at the site of the injection.Generally, the ingredients are supplied either separately or mixedtogether in unit dosage form, for example, as a dry lyophilized powderor water free concentrate in a hermetically sealed container such as anampule or sachet indicating the quantity of active agent. Where thecomposition is to be administered by infusion, it can be dispensed withan infusion bottle containing sterile pharmaceutical grade water, salineor dextrose/water. Where the composition is administered by injection,an ampule of sterile water for injection or saline can be provided sothat the ingredients may be mixed prior to administration

The rhα-Gal A (or a composition or medicament containing rhα-Gal A) isadministered by an appropriate route. In one or more embodiments, therhα-Gal A is administered systemically. In one embodiment, the rhα-Gal Ais administered intravenously. In other embodiments, rhα-Gal A isadministered by direct administration to a target tissue, such as toheart or skeletal muscle (e.g., intramuscular), or nervous system (e.g.,direct injection into the brain; intraventricularly; intrathecally).More than one route can be used concurrently, if desired.

The rhα-Gal A (or a composition or medicament containing rhα-Gal A) isadministered in a therapeutically effective amount (e.g., a dosageamount that, when administered at regular intervals, is sufficient totreat the disease, such as by ameliorating symptoms associated with thedisease, preventing or delaying the onset of the disease, and/orlessening the severity or frequency of symptoms of the disease). Theamount which will be therapeutically effective in the treatment of thedisease will depend on the nature and extent of the disease's effects,and can be determined by standard clinical techniques. In addition, invitro or in vivo assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed will also dependon the route of administration, and the seriousness of the disease, andshould be decided according to the judgment of a practitioner and eachpatient's circumstances. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.In at least one embodiment, the rhα-Gal A is administered by intravenousinfusion at a dose of about 0.1 mg/kg to about 30 mg/kg, typically about0.5 mg/kg to about 10 mg/kg. In at least one embodiment, the rhα-Gal Ais administered by intravenous infusion at a dose of about 0.5 mg/kg,about 1.0 mg/kg, about 1.5 mg/kg, about 2.0 mg/kg, about 2.5 mg/kg,about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg orabout 10 mg/kg. The effective dose for a particular individual can bevaried (e.g., increased or decreased) over time, depending on the needsof the individual. For example, in times of physical illness or stress,or if anti-α-galactosidase A antibodies become present or increase, orif disease symptoms worsen, the amount can be increased to counter thereduced effect or decrease to lessen complications caused by infusionreactions.

The therapeutically effective amount of rhα-Gal A (or composition ormedicament containing rhα-Gal A) is administered at regular intervals,depending on the nature and extent of the disease's effects, and on anongoing basis. Administration at a “regular interval,” as used herein,indicates that the therapeutically effective amount is administeredperiodically (as distinguished from a one-time dose). The interval canbe determined by standard clinical techniques. In preferred embodiments,rhα-Gal A is administered monthly, bimonthly; weekly; twice weekly; ordaily. Alternatively, rhα-Gal A is administered with the aid of amedical device that provides near continuous or continuousadministration through any compatible route. The administration intervalfor a single individual need not be a fixed interval, but can be variedover time, depending on the needs of the individual. For example, intimes of physical illness or stress, if anti-recombinant humangalactosidase A antibodies become present or increase, or if diseasesymptoms worsen, the interval between doses can be decreased or decreaseto improve efficacy or reduce infusion reactions. In some embodiments, atherapeutically effective amount of 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.5,2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 10, 15, 20 or 30 mg enzyme/kg bodyweight is administered twice a week, weekly or every other week with orwithout a pharmacological chaperone.

The rhα-Gal A may be prepared for later use, such as in a unit dose vialor syringe, or in a bottle or bag or medical device for intravenousadministration. Kits containing the rhα-Gal A, as well as optionalexcipients or other active ingredients, such as chaperones or otherdrugs, may be enclosed in packaging material and accompanied byinstructions for reconstitution, dilution or dosing for treating asubject in need of treatment, such as a patient having Fabry disease.

In at least one embodiment, the migalastat (or salt thereof) and rhα-GalA are administered simultaneously. In at least one embodiment, themigalastat (or salt thereof) and the rhα-Gal A are administeredsequentially. In at least one embodiment, the migalastat (or saltthereof) is administered prior to administration of the rhα-Gal A. In atleast one embodiment, the migalastat (or salt thereof) is administeredless than three hours prior to administration of the rhα-Gal A. In atleast one embodiment, the migalastat (or salt thereof) is administeredabout two hours prior to administration of the rhα-Gal A. In at leastone embodiment, the migalastat (or salt thereof) is administered lessthan two hours prior to administration of the rhα-Gal A. In at least oneembodiment, the migalastat (or salt thereof) is administered about 1.5hours prior to administration of the rhα-Gal A. In at least oneembodiment, the migalastat (or salt thereof) is administered about onehour prior to administration of the rhα-Gal A. In at least oneembodiment, the migalastat (or salt thereof) is administered from about50 minutes to about 70 minutes prior to administration of the rhα-Gal A.In at least one embodiment, the migalastat (or salt thereof) isadministered from about 55 minutes to about 65 minutes prior toadministration of the rhα-Gal A. In at least one embodiment, themigalastat (or salt thereof) is administered about 30 minutes prior toadministration of the rhα-Gal A. In at least one embodiment, themigalastat (or salt thereof) is administered from about 25 minutes toabout 35 minutes prior to administration of the rhα-Gal A. In at leastone embodiment, the migalastat (or salt thereof) is administered fromabout 27 minutes to about 33 minutes prior to administration of therhα-Gal A.

In at least one embodiment, the migalastat (or salt thereof) isadministered concurrently with administration of the rhα-Gal A. In atleast one embodiment, the migalastat (or salt thereof) is administeredwithin 20 minutes before or after administration of rhα-Gal A. In atleast one embodiment, the migalastat (or salt thereof) is administeredwithin 15 minutes before or after administration of the rhα-Gal A. In atleast one embodiment, the migalastat (or salt thereof) is administeredwithin 10 minutes before or after administration of the rhα-Gal A. In atleast one embodiment, the migalastat (or salt thereof) is administeredwithin 5 minutes before or after administration of the rhα-Gal A. In atleast one embodiment, the migalastat (or salt thereof) and rhα-Gal A areco-formulated.

In at least one embodiment, the migalastat (or salt thereof) isadministered after administration of the rhα-Gal A. In at least oneembodiment, the migalastat (or salt thereof) is administered up to 2hours after administration of the rhα-Gal A. In at least one embodiment,the migalastat (or salt thereof) is administered about 30 minutes afteradministration of the rhα-Gal A. In at least one embodiment, themigalastat (or salt thereof) is administered about one hour afteradministration of the rhα-Gal A. In at least one embodiment, themigalastat (or salt thereof) is administered about 1.5 hours afteradministration of the rhα-Gal A. In at least one embodiment, themigalastat (or salt thereof) is administered about 2 hours afteradministration of the rhα-Gal A.

Another aspect of the invention provides a kit for combination therapyof Fabry disease in a patient in need thereof. The kit includes apharmaceutically acceptable dosage form comprising migalastat, apharmaceutically acceptable dosage form comprising rhα-Gal A, andinstructions for administering the pharmaceutically acceptable dosageform comprising migalastat and the pharmaceutically acceptable dosageform comprising the rhα-Gal A to a patient in need thereof. In at leastone embodiment, the pharmaceutically acceptable dosage form comprisingmigalastat is an oral dosage form as described herein, including but notlimited to a tablet or a capsule. In at least one embodiment, thepharmaceutically acceptable dosage form comprising rhα-Gal A is asterile solution suitable for injection as described herein. In at leastone embodiment, the instructions for administering the dosage formsinclude instructions to administer the pharmaceutically acceptabledosage form comprising migalastat orally prior to administering thepharmaceutically acceptable dosage form comprising the rhα-Gal A byintravenous infusion, as described herein.

Without being bound by theory, it is believed that migalastat acts as apharmacological chaperone for the rhα-Gal A and binds to its activesite. Migalastat has been found to decrease the unfolding of rhα-Gal Aand stabilize the active conformation of the rhα-Gal A, preventingdenaturation and irreversible inactivation at the neutral pH,potentially allowing it to survive conditions in the circulation longenough to reach and be taken up by tissues. However, the binding ofmigalastat to the active site of rhα-Gal A also can result in inhibitionof the enzymatic activity of rhα-Gal A by preventing the naturalsubstrate, GL-3, from accessing the active site. It is believed thatwhen migalastat and the rhα-Gal A are administered to a patient underthe conditions described herein, the concentrations of migalastat andrhα-Gal A within the plasma and tissues are such that the rhα-Gal A isstabilized until it can be taken up into the tissues and targeted tolysosomes, but, because of the rapid clearance of migalastat, hydrolysisof GL-3 by rhα-Gal A within lysosomes is not overly inhibited by thepresence of migalastat, and the enzyme retains sufficient activity to betherapeutically useful.

All the embodiments described above may be combined. This includes inparticular embodiments relating to:

the nature of the pharmacological chaperone, for example migalastat; andthe active site for which it is specific;the dosage, route of administration of the pharmacological chaperone(e.g. migalastat) and the type of pharmaceutical composition includingthe nature of the carrier and the use of commercially availablecompositions;the nature of the drug, e.g. therapeutic protein drug product, which maybe a counterpart of an endogenous protein for which activity is reducedor absent in the subject, suitably rhα-Gal A, for example the rhα-Gal Aexpressed in CHO cells and comprising an increased content of N-glycanunits bearing one or more M6P residues when compared to a content ofN-glycan units bearing one or more M6P residues of Replagal (agalsidasealfa) and/or Fabrazyme (agalsidase beta) and/or containing less neutralglycans compared to Replagal (agalsidase alfa) and/or Fabrazyme(agalsidase beta); and suitably having an amino acid sequence as setforth in SEQ ID NO: 1, SEQ ID NO: 2 or as encoded by SEQ ID NO: 3.the number and type of N-glycan units on the rhα-Gal A, e.g.N-acetylglucosamine, N-acetylgalactosamine, galactose, fucose, sialicacid or complex N-glycans formed from combinations of these) attached tothe rhα-Gal A;the degree of phosphorylation of mannose units on the rhα-Gal A to formmono-M6P and/or bis-M6P;the dosage and route of administration (e.g. intravenous administration,especially intravenous infusion, or direct administration to the targettissue) of the replacement enzyme (rhα-Gal A) and the type offormulation including carriers and therapeutically effective amount; thedosage interval of the pharmacological chaperone (e.g. migalastat) andthe rhα-Gal A; the nature of the therapeutic response and the results ofthe combination therapy (e.g. enhanced results as compared to the effectof each therapy performed individually);the timing of the administration of the combination therapy, e.g.simultaneous administration of migalastat and the rhα-Gal A orsequential administration, for example wherein the migalastat (or saltthereof) is administered prior to the rhα-Gal A or after the rhα-Gal Aor within a certain time before or after administration of the rhα-GalA; and the nature of the patient treated (e.g. mammal such as human) andthe condition suffered by the individual (e.g. enzyme insufficiency).

Any of the embodiments in the list above may be combined with one ormore of the other embodiments in the list.

EXAMPLES

The compositions and processes of the present invention will be betterunderstood in connection with the following examples, which are intendedas an illustration only and not limiting of the scope of the invention.Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art and such changes and modificationsincluding, without limitation, those relating to the processes,formulations and/or methods of the invention may be made withoutdeparting from the spirit of the invention and the scope of the appendedclaims.

Example 1: Preparation of CHO Cell Clones Producing Rhα-Gal A with aHigh Content of Mono-M6P, Bis-M6P, and/or Sialic Acid-Bearing N-Glycans

DNA coding for wild-type human alpha-galactosidase A (α-Gal A) wascloned into a dual vector expression system for transfection intosuspension CHO-K1 cells. The two vectors used for transforming CHO cellswith DNA encoding rhα-Gal A (e.g. SEQ ID NO: 3) are shown in FIG. 3 .

For transfections, each vector was linearized with FspI, which cutswithin the Amp resistance gene. The digested DNA was purified withphenol chloroform extraction and quantified on a NanoDrop (ThermoScientific, ND2000). The linearized DNA was sent for coding sequenceconfirmation and transfections.

CHO Kl host cells were passaged at 7×10⁵ cells/ml in medium M1 (CD CHO+4mM Glutamine+1% HT Supplement) 24 hours before transfection. On the dayof transfection, the host cell cultures were sampled and counted.

During transfection, the host cell cultures were diluted to 10×10⁵cells/ml with medium M1 pre-warmed in water bath (36.5° C.) about 30 minand 5 ml of the diluted cells were added to a 50 ml spin tube and keptin a Kuhner incubator (36.5° C., 85% humidity, 6% CO₂ at 225 RPM).

12 μg of each vector (FIG. 3 ) was added into 776 μl of OptiPro™ SFM ina 50 ml spin tube. 24 μl of FreeStyle™ MAX Reagent was added into 776 μlof OptiPro™ SFM in a second 50 ml spin tube. The DNA-OptiPro SFM mixturewas added into FreeStyle MAX-OptiPro SFM mixture with gentle mixing, andincubated at room temperature for 10 minutes.

The diluted host cell cultures were removed from the Kuhner incubator,and 667 μl of DNA-Freestyle Max mixture was added into the host cellcultures. The transfected cell cultures were incubated in a Kuhnerincubator (36.5° C., 85% humidity, 6% CO₂ at 225 RPM) for 6 hours. 5 mlof fresh M1 medium was added into the transfected cell cultures.

In total six independent transfections were performed using the aboveprocedure.

Forty-eight hours after transfection, cells from each transfection wereplated in three 96-well plates at cell densities of 1-1.6×10⁶ cells/mland plating densities of 3000-4000 cells/100 pL/well in the selectivemedium M2 (Medium M1+9 μg/ml Blasticidin+400 μg/ml Zeocin). The 96-wellplates were incubated in a CO₂ incubator (36.5° C., 6% CO₂).

Three or four days after plating, 120 μl of fresh selective medium M2was added to each well of the 96-well plates. Over the following 2-3weeks, 150 μl of cell supernatant was replaced with equal volume offresh selective medium every 3 or 4 days.

To determine which of the six stable CHO pools would provide the bestproduct attributes, the pools were compared for cell culture viability,expression of rhα-Gal A and binding to the CIMPR. Approximately 16-18days after plating, when most of the wells reached >70% confluency, 150μl of cell supernatant was replaced with equal volume of fresh selectivemedium M2 24 hours prior to the expression analysis.

The cell pools were expanded in a pilot culture to investigate theviability over an extended period of time. The top 9 minipools withhighest enzyme activity from each transfection were selected andexpanded from 96- to 24-, to 6-well plates and into spin tubes for batchrefeeding screening with selective medium M2. Vials were frozen down(Cryopreservation Medium: 90% M1+10% DMSO) during minipool expansion.Frozen vials were placed in cryobox freezing containers in −80° C.freezer overnight and stored in liquid nitrogen tanks.

When the T9 pools were recovered, N-1 cell cultures were inoculated forbatch refeeding cultures by 1:5 dilution into fresh production medium M3(CDM4+4 mM Glutamine+1% HT Supplement) to obtain a seeding density of3.8-5.7×10⁵ cells/ml in spin tubes (day 0). The pool batch refeedingcultures (20 ml) were incubated in a Kuhner shaker (35° C., 85%humidity, 6% CO₂, and 225 RPM). Starting from day 3, cells were spundown (300 RPM, 5 min, 25° C.) and 80% supernatant was exchanged withfresh medium. When viable cell density was above 20×10⁶ cells/ml,certain amount of cells was discarded according to their growth totarget viable cells density between 25×10⁶ and 30×10⁶ cells/ml on thenext day. Pool batch refeeding cultures were harvested on day 20 bycentrifugation (3000 RPM, 10 min, 4° C.). During 18 days of culture, theviability of each cell pool was greater than 90% and the density ofviable cells was about 20-30×10⁶ cells/ml. (FIG. 4 ).

The expression of rhα-Gal A and binding to the CIMPR were assessed by4-methylumbelliferyl-α-D-galactoside enzyme assay (4MU) and binding to acolumn with immobilized cation independent mannose-6-phosphate receptor(CIMPR), respectively. FIG. 5 shows that pool number 2 maintained thehighest level of rhα-Gal A expression (as measured by enzyme activity)out to at least 16 days of cell culture.

A desirable product attribute is high binding to the CIMPR. Binding toCIMPR presents a pathway for enzyme uptake into the lysosome whichrepresents a major reservoir of accumulated substrate. The ability ofthe enzyme candidates to bind in vivo to the correct receptor forinternalization was estimated by applying the cell supernatant from eachpool to a CIMPR column (FIGS. 6A-6F). Pooled culture supernatant fromdays 15 to 17 was loaded onto an immobilized CIMPR affinity column.Increasing concentrations of free M6P from 0 to 1 mM, followed by a stepto 5 mM, were passed over the column to elute the bound enzyme. Thedegree of CIMPR binding for the cell pools ranged from 37% to 52% (asmeasured by activity). Pools 1 and 4 had the highest CIMPR binding with52% with pool 2 slightly less at 51%. Thus, these pools had morefavorable CIMPR binding characteristics than the other pools at the sametime point during culturing.

The supernatants were further evaluated for binding in a 96-well platebased CIMPR binding assay. In summary, the supernatants from varioustime points were diluted 1; 100, 1:1,000, and 1:10,000 in CIMPR bindingbuffer and then added to wells coated with CIMPR receptor. After 1 hourincubation at 37° C., the unbound enzyme was washed away and the boundenzyme was measured in the 4MU enzyme activity assay. The activity wasconverted to nanograms of enzyme bound and the calculated values wereplotted against the dilution. Pools 1 and 4 showed the best (compared toFabrazyme (agalsidase beta)) and most consistent binding of the 6 pools.

Based on the enzyme activity data and the CIMPR binding results, Pools 1and 4 were chosen as the top pools for further clone screening.

Pools 1 and 4 were thawed and passaged for a week. On the day ofplating, plating medium was prepared freshly, and both of pools wereplated into 384-well plates with cell density of 0.6 cell/30microliter/well. The pictures of all plates were captured on the day ofplating (D0), one day (D1) and two days (D2) after plating for thefuture clonality verification. A week after plating, 15 microliter M2medium was supplemented into each well of all 384-well plates.

Ten days after plating, all the plates were scanned again to observeclone recovery. The recovered cells were transferred from 384-wellplates to 96-well plates based on their confluency. Approximately 15days after plating, when most of the wells reached >70% confluency, 150μl of cell supernatant was replaced with equal volume of fresh selectivemedium M4 (CD CHO+4 mM Glutamine+1% HT Supplement+4 μg/mL ofBlasticidin+200 μg/mL of Zeocin) 24 hours prior to the expressionanalysis. As for the wells with high expression level, the pictures ofDO, D1 and D2 were pulled out and evaluated for clonality. A clone with1-2-4 cell stages from DO-D2 is considered to be derived from one singlecell. 22 clones from Pool 1 and 37 clones from Pool 4 with the highestexpression levels and good clonality were expanded for furtherevaluation. At this stage in the cell line development, the expressionlevels ranged from 7 to 23 μg/mL.

Considering the overall higher expression of clones from Pool 4 andbetter product quality of Pool 4 during pool screening stage, the top 10clones with the highest expression level from Pool 1 and top 26 clonesfrom Pool 4 were screened with the CIMPR binding assay. The 36 cloneswere first analyzed for enzyme activity. After titration, twoconcentrations of enzyme (50 and 100 nanomoles of 4MU released permilliliter per hour) were tested in the CIMPR binding assay.

Clones having high productivity and/or high CIMPR binding were chosenfor further evaluation. Four clones from Pool 1 and eight clones fromPool 4 were selected for batch refeeding evaluation in spin tubes.

Clones were expanded from 96- to 24- to 6-well plates and into spintubes for batch refeeding screening with medium M4 containing selectionpressure. Vials were frozen down (Cryopreservation Medium: 90% M1+10%DMSO) during clone expansion. Frozen vials were placed in cryoboxfreezing containers in −80° C. freezer overnight and stored in liquidnitrogen tanks.

When clones expanded from well-plates to spin tubes were recovered, N-1cell cultures were inoculated for batch refeeding cultures by 1:4-1:5dilution into fresh production medium M3 to obtain a seeding density of4.0-5.3×10⁵ cells/ml in spin tubes (day 0). The clone batch refeedingcultures (20 ml) were incubated in a Kuhner shaker (35° C., 85%humidity, 6% CO₂ and 225 RPM). Starting from day 3, cells were spun down(300 RPM, 5 min, 25° C.) and 80% supernatant was exchanged with fresh M3medium. When viable cell density was above 20×10⁶ cells/ml, a certainamount of cells was discarded according to their growth rate to targetviable cells density between 25×10⁶ and 30×10⁶ cells/ml on the nextclay. Pool batch refeeding cultures were harvested when viabilitydropped below 20% or on day 14 by centrifugation (3000 RPM, 10 min, 4°C.).

Supernatants from harvested cultures were measured for enzyme productionon each day beginning day 6 (D6). The CIMPR binding assay was performedwith D6 and D9 samples after titration to 50 and 100 nanomoles of 4-MUreleased per milliliter per hour. Based upon the growth characteristics,enzyme productivity, and CIMPR binding in the spin tube study, severalclones were chosen for further evaluation.

The top 4 clones, 01-003, 04-002, 04-018 and 04-023, were selected fromthe top 10 clones based upon cell growth performance, enzyme production,and CIMPR binding capacity. Bioreactor studies and additional spin tubestudies were used to further evaluate clones, and clones 01-003 and04-023 were selected as the top clones from Pools 1 and 4 for furtherdevelopment.

Example 2: Characterization of CIMPR Affinity of Rhα-Gal A

The ability of recombinant α-galactosidase A to bind to a CIMPR columnin vitro is an indicator of that recombinant enzyme's ability to bind tothe receptor in vivo and target the lysosomes. Two clones of rhα-Gal A(clone 01-003 and clone 04-023) were produced by perfusion culture andthe binding was determined by passing each enzyme over an immobilizedCIMPR affinity column (FIG. 7 ), as described above. FIGS. 7A-7Cillustrate that 90% of Fabrazyme (agalsidase beta) bound to the CIMPRcolumn, 80% of rhα-Gal A (01-003), and 96% of rhα-Gal A (04-023) boundthe CIMPR column. FIGS. 7D-7F illustrate how the expression andpurification can be varied to generate molecules that have higherbinding affinity for the CIMPR column.

Example 3: Stabilization of rhα-Gal A with Migalastat

The ability of migalastat to stabilize rhα-Gal A was investigated. Asseen in FIG. 8 and Table 1, migalastat has been found to decrease theunfolding of rhα-Gal A and stabilize the active conformation of rhα-GalA, preventing denaturation and irreversible inactivation at the neutralpH, potentially allowing it to survive conditions in the circulationlong enough to reach and be taken up by tissues.

TABLE 1 Sypro Orange Thermostability Tm ° C. Tm ° C. Tm ° C. pH 7.4 + pH7.4 + pH 7.4 + Tm ° C. 1 μM 10 μM 100 μM Tm ° C. pH 7.4 MigalastatMigalastat Migalastat pH 5.2 rhα-Gal A 48.9 49.4 54.6 58.4 57.7 (01-003)rhα-Gal A 49.9 50.3 57.0 60.0 58.3 (04-023)

Example 4: Oligosaccharide Characterization of rhα-Gal A Clones byLiquid Chromatography

The glycosylation profile of α-Gal A is very important for serumhalf-life and lysosomal targeting which in turn has a large influence onthe efficacy of the ERT. Each monomer of Replagal (agalsidase alfa) andFabrazyme (agalsidase beta) typically has three sites glycosylated. Eachglycosylation site on each α-Gal A molecule can have various types ofglycans which can lead to a very heterogeneous mixture of α-Gal Amolecules. FIG. 10 provides oligosaccharide profiles of the totalglycans found on different rhα-Gal A ERT therapies.

The glycan profiles of enzymes produced by both clones were analyzed inorder to elaborate on in vitro and in vivo characteristics that couldaffect clinical efficacy. Glycans from protein samples of each α-Gal Awere removed by peptide N-glycosidase F (PNGase) under standarddenaturing and reducing conditions. The extent of deglycosylation wasassessed on SDS-PAGE with the protein displaying a shift to lowerapparent molecular weight upon deglycosylation (FIG. 9 ). Followingrelease of the glycans from the protein backbone, the glycans arereacted with anthranilic acid to form 2-anthranilic acid derivatives,which are then separated by normal-phase chromatography on an aminocolumn.

Normal phase liquid chromatography on amino columns is a valuabletechnique to compare the relative abundance of all glycan forms. Theretention time is influenced by charge density and overalloligosaccharide size. Glycans eluting between 15-30 minutes are neutralglycan consisting of high-mannose and asialyo-complex oligosaccharides.

FIG. 10 is representative of the analysis of two types of rhα-Gal Aproteins designated 01-003 and 04-023. Purified glycans from differentERTs were compared in FIG. 10A-F to determine the glycan structuresfound on each ERT.

Proteins containing neutral glycan consisting of high-mannose andasialyo-complex oligosaccharides are quickly cleared from circulation byeither the mannose receptor or the asialoglycoprotein receptor.Therefore it is beneficial to have the minimal high-mannose andasialyo-complex oligosaccharides on a recombinant human α-Gal A.

Another potential glycan type is phosphorylated oligosaccharides. Theseglycans are critical for delivery of the ERT to the lysosome via theCIMPR. A phosphorylated oligosaccharide can contain either one M6Presidue (mono-M6P) or two M6P residues (bis-M6P). The bis-phosphorylatedglycan is preferred since its affinity is ˜3,000x greater for the CIMPRthan a mono-phosphorylated. Mono-phosphorylated glycans are indicated byblue stars while bis-phosphorylated glycans elute between 110 and 120minutes. Mono-phosphorylated glycans can be formed by either incompletephosphorylation or dephosphorylation by acid phosphatases released intothe conditioned media from cell death during manufacturing.

FIG. 10A shows glycans from Fabrazyme (agalsidase beta). Neutral glycans(elution 15-30 mins) account for ˜13% of the total glycans andbis-phosphorylated glycan (elution 110-120 mins) account for ˜9% of thetotal glycans. There are significant amounts of mono-phosphorylatedglycans present indicating partial phosphorylation or significantphosphatase degradation.

FIG. 10B shows glycans from Fabry ERT rhα-Gal A 04-023 (produced by cellline 04-023). Neutral glycans account for ˜ 33% of the total glycans andbis-phosphorylated glycan is higher (11%) than Fabrazyme (agalsidasebeta).

FIG. 10C shows glycans from Fabry ERT rhα-Gal A 01-003 (produced by cellline 01-003). A very low amount of neutral glycans are present (6%)which suggests that there should be reduced clearance via mannose andasialoglycoprotein receptors. However this prep contained very lowamounts of bis-phosphorylated glycans (6%) which can potentially have anegative impact on lysosomal delivery via the bloodstream.

FIGS. 10D-10F illustrate how the expression and purification can bevaried to generate molecules that have both low levels of neutralglycans (1.5-5%) and high levels of bis-phosphorylated oligosaccharides(9%-14%). By carefully monitoring the glycan map during ERT selection,expression and purification, rhα-Gal A product characteristics can bevaried to minimize off-target clearance via the mannose andasialoglycoprotein receptors while maximizing productive high affinitytargeting to the lysosome via the CIMPR.

A summary of the glycan distribution for the ERTs from 10A-10F isprovided in FIG. 11 .

A detailed comparison of the glycans for Replagal (agalsidase alfa),Fabrazyme (agalsidase beta), rhα-Gal A 04-023 and rhα-Gal A 01-003 isprovided in Tables 2-4 below. Table 2 provides a summary of amonosaccharide analysis, Table 3 provide a summary of the glycan“families”, and Table 4 provides a summary of the glycan structures.

TABLE 2 Mannose 6-Phosphate Sialic Acid (mol M6P/ (mol SA/ Alpha-Gal Amol protein) mol protein) Replagal (agalsidase alfa) 1.8 4.9 Fabrazyme(agalsidase beta) 3.4 4.8 rhα-Gal A (04-023) 3.7 4.5 rhα-Gal A (01-003)3.8 5.6

TABLE 3 ¹Replagal ²Fabrazyme ¹04-023 ²01-003 (Agalsidase (Agalsidaserhα- rhα- alfa) beta) Gal A Gal A % Area % Area % Area % Area NeutralComplex and High 24.5 12.9 17.8 6.5 Mannose Sialylated Complex 45.2 42.035.8 48.2 Hybrid 1.5 10.6  0.2 6.4 Phosphorylated Mono-M6P 1.5 10.6  0.26.4 (Hybrid) Bis-M6P Not 1.2 Not 3.0 (Hybrid) determined determinedMono-M6P 25.7 20.7 31.9 20.8 (High Mannose) Bis-M6P 3.1 6.8 14.5 11.1(High Mannose) Covered Mono-M6P Not 3.4 Not 1.8 (High Mannose)determined determined Covered Bis-M6P Not 0.1 Not 0.0 (High Mannose)determined determined ¹Glycan values derived from HPLC analysis of 2AAlabeled glycans ²Glycan values derived from LC/MS/MS method

TABLE 4 ¹Replagal ²Fabrazyme ¹04-023 ²01-003 (Agalsidase alfa)(Agalsidase beta) rhα-Gal A rhα-Gal A % Area % Area % Area % AreaNeutral (High Mannose 24.5 12.9 17.8 6.5 and Complex) Mono-Sialylated20.7 5.7 11.6 10.1 (Complex) Mono- or Di-Sialylated 2.4 0 3.4 0(Complex) Di-Sialylated (Complex) 13.0 19.3 6.9 22.7 Tri-Sialylated(Complex) 8.5 13.1 11.8 10.6 Tetra-Sialylated 0.7 3.9 1.6 4.8 (Complex)Mono-M6P; Mono- 1.5 10.6 0.2 6.4 Sialylated (Hybrid) Mono-M6P (High 25.720.7 31.9 20.8 Mannose) Bis-M6P (High 3.1 6.8 14.5 11.1 Mannose) Bis-M6P(Hybrid) Not determined 1.2 Not determined 3.1 Covered Bis-M6P Notdetermined 0.1 Not determined 0.0 Covered Mono-M6P Not determined 3.3Not determined 1.8 ¹Glycan values derived from HPLC analysis of 2AAlabeled glycans ²Glycan values derived from LC/MS/MS method

As can be seen from Tables 2-4, rhα-Gal A 01-003 and rhα-Gal A 04-023have certain distinguishing characteristics that are structurallydistinct from Replagal (agalsidase alfa) and Fabrazyme (agalsidasebeta). For example, rhα-Gal A 04-023 has very high bis-M6P content, withover 14% of glycans bearing bis-M6P. rhα-Gal A 04-023 also has highlevels of mono-M6P, with over 30% of glycans being mono-M6P (highmannose). rhα-Gal A 04-023 also has over 35% of its glycans bearingsialic acid.

Table 5 provides the relative abundancies of individual glycans ofrhα-Gal A 01-003 derived from LC/MS/MS method, and Table 6 provides asummary of the glycan structures. In Table 5 below, “MX” represents Xnumber of mannose units, “AX” represents X number of N-acetlyglucosamineantennae in a complex or hybrid glycan, “GX” represents X number ofgalactose units, “SX” represents X number of terminal sialic acid units,“F” represents a fucose unit, “P” represents a mono-M6P unit, “2(P)”represents a bis-M6P unit, “Ac” represents an acetyl unit, “X(Ac)”represents X number of acetyl caps, “KX” represents X number ofdeaminoneuraminic acid units and “SgX” represents an X number ofN-glycolylneuraminic acid units.

TABLE 5 Glycan Name Glycan % of FLD Total A1G1 0.19 A1G1S1 0.37 A2G1S10.29 A2G2 0.37 A2G2S1 1.67 A2G2S1Sg1 0.26 A2G2S2 5.09 A2G2Sg1 0.06A3G3S2 0.16 A3G3S3 0.30 A4G4S3 0.04 FA1 0.20 FA1G1 0.23 FA1G1S1 0.55FA2G1 0.52 FA2G1S1 0.87 FA2G2 0.66 FA2G2 2(Ac)S2 0.06 FA2G2AcS1 0.04FA2G2AcS2 0.13 FA2G2S1 4.31 FA2G2S1Sg1 0.70 FA2G2S2 14.99 FA2G2Sg1 0.19FA3G1S1 0.08 FA3G2 0.04 FA3G2S1 0.16 FA3G2S1K1 0.00 FA3G2S2 0.35 FA3G30.11 FA3G3S1 0.47 FA3G3S1Sg1 0.04 FA3G3S2 1.59 FA3G3S3 8.35 FA4G3S2 0.04FA4G3S3 0.14 FA4G4S2 0.19 FA4G4S2Sg1 0.00 FA4G4S3 1.16 FA4G4S3Sg1 0.43FA4G4S4 4.70 FA5G5S3 0.14 FA5G5S4 0.04 A1 2(P)M5 3.01 A1G1M5 0.21A2G1K1/A1G1S1M4 0.77 A1G1S1M5 0.72 A1G1S1PM6 4.67 A1G1Sg1M4 0.03A1G1Sg1M5 0.02 A1PM6 0.00 A1PM7 1.72 FA1G1S1M4 0.05 FA1G1S1M5 0.06FA1G1S1PM6 0.00 FA2G2S2M4 0.10 2(P)M7 10.17 2(P)M8 0.98 CPM6 1.48 CPM70.35 M4 0.29 M5 2.19 M6 0.63 M7 0.27 M8 0.21 M9 0.33 PM5 1.60 PM6 9.22PM7 9.91 PM8 0.07

TABLE 6 Total Mono-M6P (% of total glycans) 29.0 Total Bis-M6P (% oftotal glycans) 14.2 Total M6P (% of total glycans) 43.2 Total M6P(mol/mol protein) 3.4 Total Sialic (% of total glycans) 57.7 TotalSialic Acid (mol/mol protein) 7.0 Total Neutral (% of total glycans) 6.5

The rhα-Gal A 01-003 tested and shown in Tables 2-6 above has highbis-M6P (>14%), high mono-M6P (>25%), and high sialic acid (>50%) andlow neutral glycans (<7%). The rhα-Gal A 01-003 tested also has highamounts of M6P (>3 mol/mol) and sialic acid residues (>5 mol/mol) perrhα-Gal A protein.

These combinations of specific glycosylation features provide uniquerhα-Gal A enzymes that balance phosphorylation, sialylation and neutralglycan content, thus providing novel enzymes that can have enhancedtargeting and less non-productive clearance than commercially availableenzymes.

Example 5: Pharmacokinetics of Rhα-Gal a and Migalastat Combination

The pharmacokinetic profiles of rhα-Gal A 01-003 and rhα-Gal A 04-023were compared, as well as the effect of combination with migalastat(FIG. 12 .) Enzyme (1 mg/kg) was administered via an intravenous bolusinjection into Gla KO mice with or without co-formulation with 3 mg/kgmigalastat. The Gla knockout (KO) mouse model of Fabry disease shows ahigh level of analogy to the pathophysiological processes of the diseasein Fabry patients. Plasma enzyme activity was assessed at 5, 15, and 30minutes post infusion as well as 1, 2, and 24 hours post infusion. Thetreatment groups are shown in Table 7.

TABLE 7 Dose Group Treatment (mg/kg) 1 Untreated — 2 Fabrazyme(agalsidase beta) 1 3 Clone 01-003 1 4 Clone 04-023 1 5 Clone 01-003 +Migalastat 1 + 3 6 Clone 04-023 + Migalastat 1 + 3

FIG. 12 shows the pharmacokinetics of rhα-Gal A 01-003 and rhα-Gal A04-023. rhα-Gal A 04-023 has a lower pharmacokinetic profile compared toFabrazyme (agalsidase beta),while rhα-Gal A 01-003 showed a higher AUCover 2 hours, indicating a greater overall exposure to this enzymecompared to the other enzymes. FIG. 12 also shows the activity ofrhα-Gal A 01-003 in plasma is enhanced by co-formulation withmigalastat. Similarly, the activity of rhα-Gal A 04-023 in plasma isincreased by the presence of migalastat.

Table 8 shows the respective half-life and area under the curve (AUC)for Fabrazyme (agalsidase beta), rhα-Gal A 01-003 and rhα-Gal A 04-023in Gla KO mice. The level of tissue uptake of the recombinant enzymeswas assessed at 24 hours using the enzyme activity assay.

TABLE 8 rhα-Gal rhα-Gal Fabrazyme A 01-003 A 04-023 (agalsidase ERT+mig- ERT +mig- beta) alone alastat alone alastat Half-life (hr) 0.140.24 0.54 0.07 0.17 AUC_(0-2 hr) 4985 9301 24431 2545 8994 (nmol 4MU/mL/hr * hr)

FIG. 13 A shows that enzyme activity in heart tissue after 24 hours for01-003 was similar to that of Fabrazyme (agalsidase beta) whereas theactivity of rhα-Gal A 04-023 was slightly less. The activity in heartafter 24 hours for both rhα-Gal A 01-003 and rhα-Gal A 04-023 wasincreased by the presence of migalastat. The level of enzyme activityfound in strain-matched wild type mice are included as a comparator.Interestingly, after bolus infusion of recombinant enzyme the activitylevels after 24 hours are higher than those seen in wild-type hearttissue. FIG. 13B shows that the enzyme activity after 24 hour in kidneywas similar for all three enzymes and both rhα-Gal A 01-003 and rhα-GalA 04-023 were increased by the presence of migalastat with rhα-Gal A01-003 approaching wild-type levels. FIG. 13C shows that the activity inskin tissue after 24 hours was similar across all three enzymes testedand did not appear to be affected by the presence of migalastat.

Example 6: Single Administration Efficacy Comparison of Rhα-Gal aVariants

The ability of rhα-Gal A 01-003 and rhα-Gal A 04-023 to reduce plasmalyso-Gb3 and tissue GL-3 was assessed in Gla KO mice. The effect onsubstrate reduction when combining these enzymes in a co-formulationwith migalastat was also assessed. Mice were given a dose of 1 mg/kgenzyme with or without migalastat and plasma lyso-Gb3 and tissue GL-3(heart, kidney, and skin) was assessed seven days after enzymeadministration (Table 9).

TABLE 9 Group Treatment Dose (mg/kg) 1 Untreated — 2 Fabrazyme(agalsidase beta) 1 3 rhα-Gal A 01-003 1 4 rhα-Gal A 01-003 + Migalastat1 + 3 5 rhα-Gal A 04-023 1 6 rhα-Gal A 04-023 3 7 rhα-Gal A 04-023 +Migalastat 1 + 3

FIG. 14A shows the tissue enzyme activity in Gla KO mouse heart sevendays after administration of enzyme. In heart, administration of rhα-GalA 01-003 leads to similar enzyme activity after seven days asadministration of Fabrazyme (agalsidase beta) whereas addition ofmigalastat to rhα-Gal A 01-003 significantly increased the enzymeactivity level. All three enzymes gave similar activity in the kidneyafter seven days (FIG. 14B). The addition of migalastat to rhα-Gal A01-003 significantly increased the enzyme level in kidney as didincreasing the dose to 3 mg/kg (higher dose for rhα-Gal A 04-023). Inskin, after seven days the enzyme activity levels were similar forrhα-Gal A 04-023 and Fabrazyme (agalsidase beta) but was higher forrhα-Gal A 01-003 with, or without, migalastat (FIG. 14C). Increasing thedose of rhα-Gal A 04-023 lead to a higher level of enzyme activity afterseven days in skin (FIG. 15C). Higher enzyme levels were observed byaddition of migalastat to rhα-Gal A 04-023 or by increasing the dose to3 mg/kg (FIG. 14D).

In addition to assessment of enzyme activity in tissues, the degree ofsubstrate reduction seven days post-administration was determined. Inheart tissue, compared to 1 mg/mg of Fabrazyme (agalsidase beta),inclusion of migalastat significantly enhanced reduction of GL-3 (forrhα-Gal A 01-003 and rhα-Gal A 04-023) as did increasing the dose from 1mg/kg to 3 mg/kg (rhα-Gal A 04-023) (FIG. 15A). In kidney (FIG. 15B),all enzyme treatments lead to a similar reduction in GL-3 except thehigher dose of 3 mg/kg rhα-Gal A 04-023 which enhanced substratereduction.

In skin (FIG. 15C), rhα-Gal A 04-023 co-formulated with migalastat leadsto reduction in GL-3 that was significantly lower than Fabrazyme(agalsidase beta) alone. A dose of 3 mg/kg lead to a significantly lowerlevel of GL-3 in skin when compared to Fabrazyme (agalsidase beta) alone(1 mg/kg). rhα-Gal A 01-003 (1 mg/kg) co-formulated with migalastat leadto a reduction in lyso-Gb3 that was significantly lower than Fabrazyme(agalsidase beta) alone (1 mg/kg). A dose of rhα-Gal A 04-023 (3 mg/kg)resulted in significantly lower levels of plasma lyso-Gb3 than Fabrazyme(agalsidase beta) alone (1 mg/kg) (FIG. 16D).

In this Example 6, the half-life of rhα-Gal A 01-003 in Gla KO mice wasapproximately 14 minutes compared to approx. 8 minutes for Fabrazyme(agalsidase beta), and 4 minutes for rhα-Gal A 04-023 while the additionof migalastat improved the exposure of both rhα-Gal A clones.

Based on the pharmacokinetic and efficacy data, rhα-Gal A 01-003 wasselected for further development.

Example 7: Variations in Production of Rhα-Gal A

Two different studies compared the pharmacokinetics of rhα-Gal Aproduced by different processes. Gla KO mice (n=5-7/group) were given asingle IV bolus injection of 1 mg/kg of Fabrazyme (agalsidase beta) orrhα-Gal A 01-003 produced by different processes. Plasma collected fromserial bleed at 5, 15, 30, 60 and 120 min and pharmacokinetics weredetermined as measured by α-Gal A activity. The pharmacokineticproperties of Fabrazyme (agalsidase beta) and rhα-Gal A 01-003 areprovided in FIGS. 16A-16B and Tables 10 and 11. The rhα-Gal A shown inFIGS. 16A and Table 10 corresponds to the preparation shown in FIG. 10C,and the rhα-Gal A shown in FIGS. 16B and Table 11 corresponds to thepreparation shown in FIG. 10F.

TABLE 10 Fabrazyme (agalsidase rhα-Gal beta) A 01-003 T_(1/2) ^(a)(mins) 7.7 13.5 AUC_(0-2 hr) (nmol/mL/hr*hr) 4985 9301

TABLE 11 Fabrazyme (agalsidase rhα-Gal beta) A 01-003 T_(1/2) ^(a)(mins) 9.0 9.7 AUC_(0-2 hr) (nmol/mL/hr*hr) 6417 8398 T_(1/2) ^(a)determined with an upper λz of 5 min. and lower λz of 30 min.

As can be seen from FIG. 16A and Table 10, rhα-Gal A 01-003 producedaccording to a first process had a significantly longer half-life thanFabrazyme (agalsidase beta). As can be seen from FIG. 16B and Table 11,the second process produced a version of rhα-Gal A 01-003 that has asimilar apparent half-life as Fabrazyme (agalsidase beta). However, thisreduction in half-life is believed to be due to increased uptake intocells due to an increased M6P content, rather than an increase innon-productive clearance. As shown in FIGS. 10C and 10F, bothpreparations of rhα-Gal A had a low content of neutral glycans (peaks onleft of figures) and the preparation of rhα-Gal A in FIG. 10F had ahigher bis-M6P content than the preparation of rhα-Gal A in FIG. 10C(peak on right of figures). Accordingly, these studies show that evensimilar half-lives for ERTs do not necessarily demonstrate a similar ERTproduct, as the ERT can be cleared due to targeting or by non-productiveclearance.

The rhα-Gal A 01-003 produced by a variation of the manufacturingprocess was able to reduce α-Gal A substrate in heart and kidney and wasenhanced by co-formulation with migalastat (FIGS. 17A and 17B). Gla KOmice were administered 1 mg/kg of enzyme IV and GL-3 measured one weekpost-administration. Co-formulation with migalastat leads to greatersubstrate reduction than either Fabrazyme (agalsidase beta) alone orrhα-Gal A 01-003 alone.

Example 8: Pharmacokinetics of Various Doses of Rhα-Gal a with andwithout Migalastat

The pharmacokinetics of various doses of rhα-Gal A 01-003 with andwithout migalastat were compared to Fabrazyme (agalsidase beta). Enzymewas administered via a single bolus infusion into Gla KO mice (n=5; 40in total; ˜6 months old) with or without co-formulation with migalastat.The treatment groups are shown in Table 12.

TABLE 12 Group Treatment 1 Vehicle 2 Fabrazyme (agalsidase beta) 1 mg/kg3 Fabrazyme (agalsidase beta) 10 mg/kg 4 rhα-Gal 01-003 A 1 mg/kg 5rhα-Gal 01-003 A 3 mg/kg 6 rhα-Gal01-003 A 10 mg/kg 7 Co-formulation ofrhα-Gal A 01-003 1 mg/kg + Migalastat 3 mg/kg 8 Co-formulation ofrhα-Gal A 01-003 3 mg/kg + Migalastat 10 mg/kg

Serial submandibular bleedings were taken for plasma at 5 min, 15 min,30 min, 1 hr, and 2 hr. Necropsy was performed at 24 hours, and plasmaand tissues were collected.

FIGS. 18A-18F show the pharmacokinetics of the various doses of rhα-GalA with and without co-formulation with migalastat, as well as the twodoses of Fabrazyme (agalsidase beta). Table 13 below shows therespective half-life for the same treatment groups. FIGS. 18A and 18Bshow the pharmacokinetics of all of the treatment groups. FIG. 18Cfocuses on the enzyme dose of 1 mg/kg for Fabrazyme (agalsidase beta),rhα-Gal A alone and rhα-Gal A with 3 mg/kg migalastat. FIG. 18D focuseson the enzyme dose of 3 mg/kg for rhα-Gal A alone and rhα-Gal A with 10mg/kg migalastat. FIG. 18E focuses on the enzyme dose of 10 mg/kg forFabrazyme (agalsidase beta) and rhα-Gal A. FIG. 18F focuses on thepharmacokinetics of Fabrazyme (agalsidase beta) and rhα-Gal A alone atvarious enzyme doses.

TABLE 13 Fabrazyme t_(1/2) (hr) (agalsidase (95% CI) beta) rhα-Gal A 1mg/kg 0.14 0.19 (0.12, 0.16) (0.16, 0.23) 1 + 3 mg/kg migalastat 0.44(0.40, 0.48) 3 mg/kg 0.26 (0.22, 0.30) 3 + 10 mg/kg migalastat 0.49(0.40, 0.63) 10 mg/kg 0.48 0.61 (0.40, 0.60) (0.47, 0.85) Note:Half-life by one-phase decay in Prism GraphPad with no weighting

As can be seen from Table 13 and FIGS. 18A-18F, the half-life of eachenzyme increased with increasing dose. The rhα-Gal A had dose-dependentand non-linear pharmacokinetics. At the same dose, rhα-Gal A shows alonger half-life than Fabrazyme (agalsidase beta). The co-formulation ofmigalastat with rhα-Gal A also substantially increased the half-life ofenzyme activity in circulation, with up to a 2.3-fold increase in thecirculating half-life of rhα-Gal A after co-formulation with migalastat.

Example 9: Repeat Administration Efficacy Study in Gla KO Mice

The efficacy of rhα-Gal A 01-003 with and without migalastat andFabrazyme (agalsidase beta) were compared. Enzyme was administered via abolus infusion into Gla KO mice with or without co-formulation with 3mg/kg migalastat. ˜16 week old male Gla KO mice (n=8; 72 in total) weregiven two bi-weekly IV administrations. The treatment groups are shownin Table 14 and the results are shown in FIGS. 19A-19D and 20A-20C.

TABLE 14 Group number Treatment 1 Vehicle 2 Fabrazyme (agalsidase beta)1 mg/kg 3 Fabrazyme (agalsidase beta) 10 mg/kg 4 rhα-Gal A 1 mg/kg 5Co-formulation of rhα-Gal A 1 mg/kg + migalastat 3 mg/kg 6Co-formulation of rhα-Gal A 1 mg/kg + migalastat 10 mg/kg 7 rhα-Gal A 3mg/kg 8 Co-formulation of rhα-Gal A 3 mg/kg + migalastat 3 mg/kg 9Co-formulation of rhα-Gal A 10 mg/kg + migalastat 10 mg/kg

As can be seen from FIG. 19A (heart) and FIG. 19B (kidney), a gooddose-response seen for rhα-Gal A and Fabrazyme (agalsidase beta) alone:10 mg/kg Fabrazyme (agalsidase beta) >3 mg/kg rhα-Gal A>1 mg/kg rhα-GalA=1 mg/kg Fabrazyme (agalsidase beta). Co-formulation of 1 mg/kg rhα-GalA+3 or 10 mg/kg migalastat show significant reduction in GL-3 levelscompared to 1 mg/kg Fabrazyme (agalsidase beta). 3 mg/kg rhα-Gal Aco-formulated with 3 or 10 mg/kg migalastat achieved similar or evenslightly better GL-3 reduction compared to 10 mg/kg Fabrazyme(agalsidase beta), with 10 mg/kg migalastat co-form appearing to deliverthe best results.

As can be seen from FIG. 19C (skin) and FIG. 19D (plasma lyso-GB3), agood dose-response seen for rhα-Gal A and Fabrazyme (agalsidase beta)alone: 10 mg/kg Fabrazyme (agalsidase beta)>3 mg/kg rhα-Gal A>1 mg/kgrhα-Gal A≈1 mg/kg Fabrazyme (agalsidase beta). In 1 mg/kg rhα-Gal Agroup there was high variability (with one likely outlier). Overall, asimilar observation to heart and kidney. Co-formulation of 1 mg/kgrhα-Gal A+3 or 10 mg/kg migalastat shows significant lower GL-3 levelsthan 1 mg/kg Fabrazyme (agalsidase beta). 3 mg/kg rhα-Gal Aco-formulated with 3 or 10 mg/kg migalastat achieved similar or evenslightly better GL-3 reduction compared to 10 mg/kg Fabrazyme(agalsidase beta), with 10 mg/kg migalastat co-form appearing to deliverthe best results.

Example 10: Single Administration Efficacy Comparison of Various Dosesof Rhα-Gal A

The efficacy of rhα-Gal A 01-003 with and without migalastat andFabrazyme (agalsidase beta) were compared. Enzyme was administered via asingle bolus infusion into Gla KO mice with or without co-formulationwith migalastat. ˜18 week old male Gla KO mice (7 per group) were givena single bolus IV administration and necropsies were performed 7 daysafter dose. The treatment groups are shown in Table 15 and the resultsare shown in FIGS. 21A-21D and Tables 16-19.

TABLE 15 Group number Treatment  1 Vehicle  2 Fabrazyme (agalsidasebeta) 1 mg/kg  3 Fabrazyme (agalsidase beta) 10 mg/kg  4 Fabrazyme(agalsidase beta) 25 mg/kg  5 Co-formulation of rhα-Gal A 1 mg/kg +migalastat 3 mg/kg  6 Co-formulation of rhα-Gal A 3 mg/kg + migalastat10 mg/kg  7 rhα-Gal A 10 mg/kg  8 Co-formulation of rhα-Gal A 10 mg/kg +migalastat 3 mg/kg  9 Co-formulation of rhα-Gal A 10 mg/kg + migalastat30 mg/kg 10 Co-formulation of rhα-Gal A 10 mg/kg + migalastat 300 mg/kg

TABLE 16 % reduction % reduction % reduction Heart compared to Kidneycompared to Skin compared to (μg/g Fabrazyme (μg/g Fabrazyme (μg/gFabrazyme GL-3 levels tissue) (1 mg/kg) tissue) (1 mg/kg) tissue) (1mg/kg) Fabrazyme — 183.23 443 1465 1 mg/kg — rhα-Gal A 1 mg/kg + 91.4050.12 340 23.25 1300 11.26 migalastat 3 mg/kg — rhα-Gal A 3 mg/kg +38.10 79.21 254 42.66 219 85.05 migalastat 10 mg/kg

TABLE 17 % reduction % reduction % reduction Heart compared to Kidneycompared to Skin compared to (μg/g Fabrazyme (μg/g Fabrazyme (μg/gFabrazyme GL-3 levels tissue) (10 mg/kg) tissue) (10 mg/kg) tissue) (10mg/kg) Fabrazyme — 53.20 281 207 10 mg/kg rhα-Gal A — 30.30 43.05 2771.42 149 28.02 10 mg/kg — rhα-Gal A 10 mg/kg + 18.10 65.98 203 27.76 10449.76 migalastat 3 mg/kg — rhα-Gal A 10 mg/kg + 19.30 63.72 202 28.1176.80 62.90 migalastat 30 mg/kg — rhα-Gal A 10 mg/kg + 31.10 41.54 21922.06 124 40.10 migalastat 300 mg/kg Fabrazyme — 21.10 203 102 25 mg/kg

TABLE 18 Plasma % reduction compared to Plasma lyso-Gb₃ levels (ng/mL)Fabrazyme (1 mg/kg) Fabrazyme — 122 1 mg/kg — rhα-Gal A 1 mg/kg + 10315.57 migalastat 3 mg/kg — rhα-Gal A 3 mg/kg +  29 75.66 migalastat 10mg/kg

TABLE 19 Plasma % reduction compared to Plasma lyso-Gb₃ levels (ng/mL)Fabrazyme (10 mg/kg) Fabrazyme — 29.5 10 mg/kg rhα-Gal A — 28.7  2.71 10mg/kg — rhα-Gal A 10 mg/kg + 17.6 40.34 migalastat 3 mg/kg — rhα-Gal A10 mg/kg + 12.6 57.29 migalastat 30 mg/kg — rhα-Gal A 10 mg/kg + 18.337.97 migalastat 300 mg/kg Fabrazyme — 18.1 25 mg/kg

As can be seen from FIGS. 21A-21D and Tables 16-19, the co-formulationof rhα-Gal A 1 mg/kg+migalastat 3 mg/kg had a greater reduction insubstrate (GL-3 and lyso-Gb3) than a Fabrazyme (agalsidase beta) at adose of 1 mg/kg, and the co-formulation of rhα-Gal A 3 mg/kg+migalastat10 mg/kg was comparable to the higher dose of Fabrazyme (agalsidasebeta) of 10 mg/kg. The co-formulation of rhα-Gal A 10 mg/kg withmigalastat achieved similar efficacy as the higher dose of Fabrazyme(agalsidase beta) of 25 mg/kg, with the co-formulation of rhα-Gal A 10mg/kg+migalastat 30 mg/kg providing the greatest overall substratereduction. The rhα-Gal A dose of 10 mg/kg provided greater substratereduction than an equivalent dose of Fabrazyme (agalsidase beta) of 10mg/kg, and the co-formulation with migalastat further enhanced thesubstrate reduction.

Example 11: Intravenously Administered Migalastat Dose Escalation toEvaluate Safety, Tolerability and Pharmacokinetics

Healthy volunteers received migalastat HCl via the IV route ofadministration to evaluate safety, tolerability, and pharmacokinetics.Another study was performed to determine the absolute bioavailability ofmigalastat after an oral capsule dose as compared to an IV administereddose of migalastat HCl in healthy volunteers. Characterizing the PKbehavior of migalastat alone after IV administration was expected toallow for a more precise prediction of migalastat HCl proposed doses inco-formulation with rhα-Gal A. The absolute bioavailability arm wasadded to determine the exposure ratio of a 150 mg oral dose relative toa 150 mg dose administered as an IV infusion. This was investigated todetermine if oral to IV bridging was possible and to provide an accuratedose determination of IV migalastat HCl for co-administration and/orco-formulation with rh-Gal A. A list of the primary and secondaryobjectives and endpoints is provided in Table 20.

TABLE 20 PRIMARY Objectives Endpoints Determine the pharmacokinetics ofPlasma PK: AUC_(0-∞,) AUC_(0-t), C_(max), t_(max), migalastat HCLfollowing a single CL_(T), V_(z), and t_(1/2) for migalastat 2-hourinfusion in healthy subjects Determine the safety and tolerabilitySafety: adverse events (AEs) including of a single migalastat HCl 2-hourinfusion site reactions and allergic infusion in healthy subjectsreactions, clinically significant changes in safety laboratory tests(hematology, chemistry, and urinalysis), vital signs (blood pressure,heart rate, respiratory rate, temperature), physical examinations, and12-lead electrocardiograms (ECGs) Secondary Objectives EndpointsDetermine dose proportionality of Plasma PK: AUC_(0-∞,) AUC_(0-t), andC_(max) migalastat following a single 2-hour IV infusion in healthysubjects (at doses of 0.3, 1.0, and 10.0 mg/kg) Determine the urinaryexcretion of Urinary PK: Ae_(0-24 h), Fe, CL_(r) of the unchangedmigalastat following migalastat a single 2-hour IV infusion in healthysubjects Determine the absolute bioavail- Plasma PK: F_(abs), the pointestimate for ability of plasma migalastat AUC_(0-∞), AUC0-t, ratios and90% confidence intervals (CIs)

Type of Study: This Phase 1 study was a single-center study consistingof 2 designs. Cohorts 1, 2, and 3 were performed according to arandomized, double-blind, placebo-controlled, single ascending dose(SAD) IV study design to evaluate the safety, tolerability, and PK of IVadministered migalastat HCl or IV administered placebo in healthysubjects. Cohort 4 was an open-label, randomized two-way crossover armdesigned to assess the absolute bioavailability of orally administeredmigalastat HCl relative to IV administered migalastat HCl in healthysubjects. In all cohorts, the duration of the IV infusion withmigalastat HCl (or placebo, if applicable) was 2 hours (±10 min) at aconstant rate of 125 mL/h.

Cohorts 1, 2, 3: Subjects in Cohorts 1, 2, and 3 received IVadministered single ascending doses of migalastat HCl. Seven subjectswere enrolled into each cohort (5 subjects randomized to migalastat HCland 2 subjects randomized to placebo). Dosing of Cohorts 1, 2, and 3 wasstaggered to allow for safety and tolerability review before proceedingwith the next group. Therefore, Cohorts 1, 2, and 3 were each dividedinto 2 sub-groups: Group A (2 subjects randomized to migalastat HCl and1 subject randomized to placebo) and Group B (3 subjects randomized tomigalastat HCl and 1 subject randomized to placebo). Subjects in Group Bwere dosed the day following completion of Group A dosing. Except forCohort 3B, which was dosed 31 days after Cohort 3A because an interim PKreview was performed on emerged data from Cohort 3A. Additionally,initiation of infusion was staggered by 60 minutes for Group A subjects,and by 30 minutes for Group B subjects (see FIG. 22 ).

-   -   In all cohorts, dosing of subjects in Group B started the day        after dosing in Group A. Except for Cohort 3B which was dosed 31        days after Cohort 3A, because an interim PK review of Cohort 3A        was performed.

Cohort 4: An open-label crossover arm (Cohort 4) was added to assess theabsolute bioavailability of orally administered150 mg migalastat HClcapsule relative to 2-hour IV infusion of 150 mg migalastat HCl. Cohort4 consisted of 2 sequential treatment periods with a washout period of 7days between study drug administrations (FIG. 23 ). Cohort 4 enrolled 10subjects all of whom received active drug (no placebo was administered).Cohort 4 was randomized such that 5 subjects received the oralformulation and 5 subjects received the IV formulation during Period 1,followed by the alternate formulation during Period 2.

-   -   * During Period 1, all Day−1 through Day 3 assessments were        performed. ** The washout period was 7 days. *** During Period        2, subjects repeated all Day−1 through Day 3 assessments.

Dose Escalation: This escalating design was chosen to allow carefulincrease of the dose, after assessment of safety and tolerability ofeach preceding dose. The IV administered single ascending doses ofmigalastat HCl include a starting dose of 0.3 mg/kg (Cohort 1), amid-range dose of 1.0 mg/kg (Cohort 2), and a highest dose of permittedat any time during a cohort based on emergent safety, tolerability, andPK data, up to a maximum allowed dose of 13.0 mg/kg. As per CSPAmendment 3.1 (see Appendix 16.1.1 and Section 9.8.1), the top dose ofthis study (Cohort 3) was estimated to not exceed the following limit:The maximum exposure (AUC) following oral administration of 1250 mgmigalastat HCl of 116 h*μg/mL (115,931 h*ng/mL).

Treatments Administered: In all cohorts, the duration of the IV infusionwith migalastat HCl (or placebo, if applicable) was 2 hours (±10 min) ata constant rate of 125 mL/h. Subjects enrolled in Cohorts 1, 2, and 3were randomized to receive one of the following doses of migalastat HCl(n=5) or placebo (n=2):

Cohort 1 (0.3 mg/kg IV): migalastat HCl (n=5) or placebo (n=2)

Cohort 2 (1.0 mg/kg IV): migalastat HCl (n=5) or placebo (n=2)

Cohort 3 (10.0 mg/kg IV): migalastat HCl (n=5) or placebo (n=2).

Cohort 4: Period 1: 150 mg migalastat HCl capsule (n=5) or 150 mgmigalastat HCl via IV infusion (n=5). Period 2: 150 mg migalastat HClcapsule (n=5) or 150 mg migalastat HCl via IV infusion (n=5)

The migalastat HCl used for infusion was 50 mg/ml and the placebo was0.9% sodium chloride. The oral migalastat HCl was administered as a 150mg gelatin tablet.

Selection of Doses in the Study: The current single dose levels wereselected on the basis of predicted human PK following IV dosing,non-clinical safety margins, and target therapeutic migalastat exposuresfor efficacy. A starting single IV infusion of 0.3 mg/kg migalastat HClin humans was selected based on large safety margins (Table 21).Likewise, the migalastat HCl 10 mg/kg dose in humans was supported byprevious oral human migalastat exposures and preclinical safety margins(Table 22). Based on a mass-balance study, the recovery of migalastatwas high, indicating that migalastat is well absorbed, however,approximately 20% to 25% may represent unabsorbed drug. As such, thepredicted exposures were expected to be as much as 1.25-fold greaterfollowing IV dosing to account for possible unabsorbed drug followingoral dosing.

TABLE 21 Predicted Human Migalastat AUC and C_(max) Following Single IVDose Administration and Exposure Margins to General Toxicology NOAELsMigalastat Predicted Predicted HCl Median Fold Cover to Median FoldCover to Dose AUC NOAEL AUC C_(max) NOAEL C_(max) (mg/kg) (h · μg/mL)Rat¹ Monkey² (μg/mL) Rat¹ Monkey² 0.3 1.86 134 165 0.36 65 68 10.0 61.54.05 4.98 12.1 1.93 2.02 Abbreviations: AUC = area under theconcentration-time curve; C_(max) = maximum observed concentration; HCl= hydrochloride; NOAEL = no observed adverse effect level. ¹Rat NOAEL(1500 mg/kg/day): AUC = 249 μg · h/mL, C_(max) = 23.4 μg/mL. ²MonkeyNOAEL (500 mg/kg/day): AUC = 306 μg · h/mL, C_(max) = 24.5 μg/mL.

TABLE 22 Predicted Human Migalastat AUC and C_(max) Following Single IVDose Administration and Exposure Margins to Human Observed OralMigalastat Exposures Following a Single Oral Dose of 1250 mg PredictedFold Cover to Predicted Fold Cover to Migalastat Median AUC from humanMedian C_(max) from human HCl Dose AUC oral 1250 mg C_(max) oral 1250 mg(mg/kg) (h · μg/mL) Min¹ Median² Max³ (μg/mL) Min¹ Median² Max³ 0.3 1.8615.5 39 62 0.36 2.6 36 61 10.0 61.5 0.47 1.19 1.89 12.1 0.08 1.07 1.81Abbreiations: AUC = area under the concentration-time curve; C_(max) =maximum observed concentration; HCl = hydrochloride; Max = maximum; Min= minimum. ¹Minimum observed individual PK exposures: AUC = 28.9 h ·μg/mL, C_(max) = 4.78 μg/mL. ²Median observed individual PK exposures:AUC = 73.2 h · μg/mL, C_(max) = 13.0 μg/mL. ³Maximum observed individualPK exposures: AUC= 115.9 h · μg/mL, C_(max) = 21.9 μg/mL.

Oral administration of the 150 mg migalastat HCl capsule was welltolerated in previous clinical studies conducted in healthy volunteersand Fabry patients. Additionally, single dose administrations up to 2000mg migalastat HCl to healthy volunteers in Phase 1 studies have beenwell tolerated. Oral administrations have demonstrated linear exposuresto 1250 mg migalastat HCl. A phase 2a study conducted in 23 Fabrypatients demonstrated improved tissue uptake of active α-Gal A into skinfollowing co-administration of a single oral administration of 150 mgmigalastat HCl with 0.2, 0.5, and 1.0 mg/kg agalsidase. Additionally,oral 150 mg migalastat HCl has been safely administered every other dayto Fabry patients with amenable mutations in Phase 2 and 3 studies inthe monotherapy program.

Cohorts 1, 2, and 3 (SAD): After a single 2-hour IV infusion ofmigalastat HCl at doses in the range of 0.3 to 10 mg/kg, maximumarithmetic mean plasma concentrations of migalastat were typicallyreached at the end of infusion, corresponding to 2 h after start ofinfusion. The mean migalastat plasma concentration-time profilesdisplayed a clear dose-dependent increase in plasma concentrationsfollowing increasing IV doses of migalastat HCl. After reaching amaximum, the migalastat concentrations decreased rapidly until 12 hpost-dose. At the higher dose levels (0.3 mg/kg and 10 mg/kg), theinitial rapid decline of migalastat concentrations was followed by amore gradual decline in an apparently biphasic manner (FIGS. 24A and 24Bshow pharmacokinetics with a linear and logarithmic scale,respectively). Combined individual migalastat plasma concentration-timeprofiles showed minimal inter-individual differences in individual PKprofiles across tested IV doses.

Cohort 4: After oral administration of 150 mg migalastat HCl, migalastatappeared in plasma within 0.25 h to 0.5 h post-dose (0.25 h was thefirst post-dose sampling time point). Maximum arithmetic mean plasmaconcentrations of migalastat were typically reached between 2 h and 4 hpost oral dose. For most subjects a single peak plasma concentration wasobserved, except that for Subject 022 an initial peak concentration at1.5 h was followed by a second and higher peak concentration at 4 h postoral dose. For Subject 030 a similar plateau phase was observed between1 h and 1.5 h post oral dose, followed by a peak concentration at 3 hpost-dose. After IV administration of 150 mg migalastat HCl the maximumarithmetic mean plasma concentrations of migalastat were reached at theend of infusion (corresponding to 2 h after start of infusion), similarto the SAD data. The maximum arithmetic mean plasma concentrations wereapproximately 2-fold higher after IV dosing than after oral dosing ofmigalastat HCl. After reaching a maximum, for both oral and IVadministration of migalastat HCl the migalastat concentrations decreasedrapidly followed by a more gradual decline in an apparently biphasicmanner (FIGS. 25A and 25B show pharmacokinetics with a linear andlogarithmic scale, respectively).

Combined individual migalastat plasma concentration-time profiles showedmore inter-individual differences in individual PK profiles after oraladministration of 150 mg migalastat HCl than after IV administration of150 mg migalastat HCl. Also the inter-individual differences in PKprofiles appeared to be more pronounced after the 150 mg IV dose thanafter the 0.3 to 10 mg/kg IV dose range, probably related tointer-individual differences in the rate and extent of absorption andclearance after the 150 mg oral dose, or because the 150 mg IV dose wasnot corrected for the individual subjects' weight.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is: 1-155. (canceled)
 156. A method for treating Fabrydisease, the method comprising administering to a patient in needthereof a pharmaceutical composition comprising a human recombinantα-galactosidase A (rhα-Gal A), wherein the rhα-Gal A comprises a proteinhaving at least 98% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2,wherein the protein has less than 10% of total N-linked oligosaccharidesthat are neutral as measured by liquid chromatography.
 157. The methodof claim 156, wherein the protein has 3.5 to 6 moles ofmannose-6-phosphate (M6P) residues per mole of rhα-Gal A homodimer asmeasured by liquid chromatography.
 158. The method of claim 156, whereinthe rhα-Gal A has one or more of: at least 17% of total N-linkedoligosaccharides that contain a single sialic acid residue as measuredby liquid chromatography; at least 20% of total N-linkedoligosaccharides that contain two sialic acid residues as measured byliquid chromatography; at least 40% of total N-linked oligosaccharidesthat contain one or two sialic acid residues as measured by liquidchromatography; or at least 4 moles of sialic acid residues per mole ofrhα-Gal A homodimer as measured by liquid chromatography.
 159. Themethod of claim 156, wherein the rhα-Gal A has at least 50% of totalN-linked oligosaccharides that contain sialic acid as measured by liquidchromatography.
 160. The method of claim 156, wherein the rhα-Gal A hasat least 25% of total N-linked oligosaccharides that aremono-mannose-6-phosphate and at least 6% of total N-linkedoligosaccharides that are bis-mannose-6-phosphate as measured by liquidchromatography.
 161. The method of claim 156, wherein the rhα-Gal A hasat least 7 moles of sialic acid residues per mole of rhα-Gal A homodimeras measured by liquid chromatography.
 162. The method of claim 156,wherein the rhα-Gal A has at least 22% of total N-linkedoligosaccharides that contain two sialic acid residues as measured byliquid chromatography.
 163. The method of claim 156, wherein the rhα-GalA has at least 14% of total N-linked oligosaccharides that arebis-mannose-6-phosphate as measured by liquid chromatography.
 164. Themethod of claim 156, further comprising administering a pharmacologicalchaperone for α-Gal A.
 165. The method of claim 164, wherein thepharmacological chaperone is migalastat or salt thereof.
 166. The methodof claim 165, wherein the pharmacological chaperone is migalastathydrochloride.
 167. The method of claim 166, wherein the patient isadministered about 150 mg of migalastat hydrochloride.
 168. The methodof claim 167, wherein the migalastat hydrochloride is administeredorally.
 169. The method of claim 156, wherein the rhα-Gal A isadministered at a dose of about 0.5 mg/kg to about 10 mg/kg.
 170. Themethod of claim 166, wherein the composition comprising rhα-Gal A isadministered once a month to once a week.
 171. The method of claim 156,wherein the composition comprising rhα-Gal A is administered every otherweek.
 172. The method of claim 171, wherein the rhα-Gal A isadministered at a dose of about 0.5 mg/kg to about 10 mg/kg.
 173. Themethod of claim 156, wherein the rhα-Gal A is co-formulated with apharmacological chaperone for α-Gal A.